Background: Using genetic enhancement techniques has led to the development of poultry capable of fulfilling significant portions of human dietary requirements through meat and egg production. Maximizing genetic potential necessitates the provision of conducive environmental conditions. Heat stress poses a substantial challenge within the poultry sector, detrimentally impacting poultry production and growth, there by diminishing breeders' economic returns. Adaptation to heat stress is contingent upon a multitude of factors. The capacity to establish homeostasis through alterations in gene expression serves to mitigate extracellular damage and restore physiological equilibrium upon reversion to favorable temperature ranges. Domestic birds thrive within a temperature spectrum of 18-24 °C, with any deviation beyond this range precipitating elevated mortality rates. Elevated mortality is attributed to heat-induced suppression of the immune system and compromised resilience. Moreover, heat stress amplifies the secretion of the corticotropin-releasing hormone, subsequently elevating blood corticosterone levels, thereby instigating molecular alterations under stress conditions. Hence, a comprehensive examination of this challenge from diverse perspectives holds significant promise. Consequently, the present study endeavors to delineate protein complexes and pivotal signaling pathways regulated by distinct genes expressed in response to heat stress within the hypothalamus of broiler chickens. Methods: The systemic response of chickens' hypothalamus transcripts to heat stress was comprehensively examined in this investigation. Transcript expression profiles from eight chicken hypothalamus tissues, including four subjected to thermal stress and four under non-stress conditions, were retrieved from the GEO database (accession number GSE37400). The raw microarray data underwent rigorous quality control measures and normalization using GEO2R software to ensure reliability and consistency. Genes with discernible expression changes were identified based on a stringent criterion of P-value > 0.05 and (LogFC < 0.5-0.5). Subsequently, a gene expression regulatory network was constructed to elucidate the interplay among genes implicated in broiler heat stress, employing Cytoscape software in conjunction with the STRING database. The network visualization facilitated the identification of protein clusters, representing areas of high density, which were further analyzed using the CytoCluster plugin renowned for its efficacy in detecting such densely interconnected regions. Following the delineation and characterization of genes within these clusters, the DAVID database was utilized to elucidate the relevant biological pathways associated with the identified genes. This comprehensive approach enabled a detailed exploration of the molecular mechanisms underlying the hypothalamic response to heat stress in chickens. Results: The analysis of microarray data unveiled a comprehensive portrait of the molecular response to heat stress in chickens. Among the 43,607 initially examined probes, 593 differentially expressed genes were identified after rigorous curation involving the elimination of duplicate genes and application of a significance threshold. A network encompassing 149 genes with a minimum interaction score of 0.4 was constructed utilizing the STRING software, shedding light on the intricate interplay among these genes. Subsequent investigations honed in on genes exerting significant regulatory effects in response to heat stress, elucidating their pivotal roles in orchestrating physiological processes. Functional assessment of the networked genes revealed their involvement in diverse biological activities, including the positive regulation of T cell- mediated immunity, WNT and MAPK signaling pathways, as well as phagocytosis and natural killer (NK) cell activity. Particularly noteworthy was the observation of suppressed NK cell activity in broilers under heat stress, likely influenced by heightened corticosterone levels and subsequent inhibition of thyroid hormone secretion, thereby impacting temperature regulation and metabolism. Moreover, the elevation in blood glucose levels observed in stressed chicks may contribute to their adaptive survival response. Importantly, heat stress was observed to disrupt cellular homeostasis and molecular pathways, manifesting in alterations in biological processes such as the cell cycle. This disruption in the cell cycle may signify a novel mechanism of stress resistance in vertebrates, further underscoring the complexity of the physiological response to thermal stress in chickens. Conclusion: The analysis of gene functionality within the network underscores the intricate nature of the innate immune response and cellular demise elicited by heat stress. Notably, certain genes identified in this analysis participate in positively regulating T cell-mediated immunity, vascular endothelial growth factor (VEGF), WNT signaling, and mitogen-activated protein kinase (MAPK) pathways. Introducing a biomarker panel comprising genes, such as MAPK14, NT3, SOX2, YAP1, FGF2, LRRK1, LEF1, and TLE1, holds promise for advancing our understanding of the underlying molecular mechanisms governing stress responses in poultry. Furthermore, this approach may facilitate the discovery of novel genes whose modulation could mitigate the effects of thermal stress. [ABSTRACT FROM AUTHOR]