This study aimed to identify differentially expressed genes (DEGs) in the testes of Bactrian camels during estrus and anestrus and investigate the regulatory role of cholecystokinin (CCK) and its receptor (CCKBR) in androgen synthesis. RNA sequencing was performed on six testicular samples (estrus, n = 3; anestrus, n = 3). A total of 291 DEGs were identified, including 27 upregulated and 264 downregulated in estrus. Gene Ontology (GO) enrichment analysis revealed that CCKBR was significantly enriched in reproduction-related pathways, and STRING analysis showed a close association between CCK and CCKBR. Further qRT-PCR, Western blot, and immunofluorescence (IF) analyses demonstrated significantly higher mRNA and protein levels of CCK/CCKBR in estrus testes (P < 0.01), with both localized in Sertoli cells, Leydig cells, primary spermatocytes, and spermatogonia. Primary Sertoli cells, confirmed by WT1 co-localization, were transfected with p-IRES2-EGFP-CCK and siRNA-CCK. Results showed that CCK overexpression significantly reduced testosterone (T) and dihydrotestosterone (DHT) levels (P < 0.05), while upregulating androgen receptor (AR) and key androgen synthesis enzymes (StAR, P450scc, 3β-HSD) (P < 0.05 or P < 0.01). In contrast, siRNA-CCK exerted the opposite effects. In conclusion, our study highlights the CCK/CCKBR axis as a crucial regulator of seasonal testicular function in Bactrian camels, with CCK negatively regulating testicular androgen synthesis by modulating AR and androgen synthesis enzymes. These findings provide valuable insights into the reproductive biology of Bactrian camels and offer a novel pathway for understanding seasonal fertility regulation in male mammals. This has important implications for enhancing camel breeding efficiency and supporting conservation efforts.