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Background: Some researchers have reported that Toxoplasma gondii can cause serious reproductive impairment in male animals. Specifically, T. gondii destroy the quality of sperm in the epididymis, which affects their sexual ability. However, among such studies, none have investigated the male reproductive transcriptome. Therefore, to investigate the relationship between T. gondii and sperm maturation, we infected mice with T. gondii prugniaud (PRU) strain and performed transcriptome sequencing of the epididymis. Results: Compared with the control group, 431 upregulated and 229 downregulated differentially expressed genes (DEGs) were found (P-value < 0.05, false discovery rate (FDR) < 0.05 and |log2 (fold change)| ≥ 1). According to results of a bioinformatics analysis, Gene Ontology (GO) function is divided into three categories: cellular component, molecular function and biological process. Upon performing GO analysis, we found that some DEGs correlated with an integral part of membrane, protein complex, cell surface, ATP binding, immune system process, signal transduction and metabolic process which are responsible for the epididymal injury. DEGs were mapped to 101 unique KEGG pathways. Pathways such as cytokine-cytokine receptor interaction, glycolysis/gluconeogenesis and apoptosis are closely related to sperm quality. Moreover, Tnfsf10 and spata18 can damage the mitochondria in sperm, which decreases sperm motility and morphology. Conclusions: We sequenced the reproductive system of male mice chronically infected with T. gondii, which provides a new direction for research into male sterility caused by Toxoplasma infection. This work provides valuable information and a comprehensive database for future studies of the interaction between T. gondii infection and the male reproductive system. [ABSTRACT FROM AUTHOR]