We discovered odorous 16-androstenes (Androstenone and Androstenol) in endangered mouse deer during a captive breeding program. This study examined the molecular characteristics, their synthesis pathway, and the possible functional role of these compounds in the reproduction of mouse deer. CYP17A1 and CYB5 genes were cloned and expressed in HEK-293, COS-7 cell lines, and gonads of mouse deer to investigate the CYP17A1 gene’s andien-β-synthase activity towards the synthesis of 16-androstenes in mouse deer. An enzyme immunoassay was further developed and standardized to measure fecal androstenone during the reproductive cycles of mouse deer. Results showed that the mouse deer CYP17A1 gene possesses andien-β-synthase activity and could transform pregnenolone into 5,16-androstadien-3β-ol. The expression of the CYP17A1 gene upregulated in the testis and ovary compared to other tissues in mouse deer. Significantly elevated androstenone and estrogens were recorded prior to delivery and postpartum estrus/mating in mouse deer. Further, there were weak correlations between fecal androstenone and estrogens/androgens in mouse deer during the breeding season. These findings suggest that androstenone probably plays a role in the reproductive activities of mouse deer. This knowledge can be used for captive breeding programs of mouse deer in India and elsewhere.