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Abstract Background Culex pipiens pallens (Diptera: Culicidae) can survive at low temperature for long periods. Understanding the effects of low-temperature stress on the gut microflora and gene expression levels in Cx. pipiens pallens, as well as their correlation, will contribute to the study of the overwintering mechanism of Cx. pipiens pallens. Methods The gut bacteria were removed by antibiotic treatment, and the survival of Cx. pipiens pallens under low-temperature stress was observed and compared with the control group. Then, full-length 16S rRNA sequencing and the Illumina HiSeq X Ten sequencing platform were used to evaluate the gut microflora and gene expression levels in Cx. pipiens pallens under low-temperature stress. Results Under the low-temperature stress of 7 °C, the median survival time of Cx. pipiens pallens in the antibiotic treatment group was significantly shortened by approximately 70% compared to that in the control group. The species diversity index (Shannon, Simpson, Ace, Chao1) of Cx. pipiens pallens decreased under low-temperature stress (7 °C). Non-metric multidimensional scaling (NMDS) analysis divided all the gut samples into two groups: control group and treatment group. Pseudomonas was the dominant taxon identified in the control group, followed by Elizabethkingia and Dyadobacter; in the treatment group, Pseudomonas was the dominant taxon, followed by Aeromonas and Comamonas. Of the 2417 differentially expressed genes (DEGs), 1316 were upregulated, and 1101 were downregulated. Functional GO terms were enriched in 23 biological processes, 20 cellular components and 21 molecular functions. KEGG annotation results showed that most of these genes were related to energy metabolism-related pathways. The results of Pearson’s correlation analysis showed a significant correlation between the gut microcommunity at the genus level and several DEGs. Conclusions These results suggest that the mechanism of adaptation of Cx. pipiens pallens to low-temperature stress may be the result of interactions between the gut bacterial community and transcriptome. Graphical Abstract