Seasonal reproduction is a strategy for mammals to adapt to environmental changes. Daurian ground squirrel (Spermophilus dauricus) is a hibernating species and a typical seasonal breeder that mates and reproduces only once a year after emerging from hibernation in the wild. Under laboratory conditions, the artificial mating and breeding technology after hibernation is still immature, and this species has not yet been domesticated as a laboratory animal. Vitamin A (VA) can be metabolized into retinoic acid (RA) within mammalian organisms. Recent research has revealed that RA promotes the differentiation of spermatogonia, the meiotic division of spermatocytes, and the transformation of round spermatids into mature spermatozoa. To explore whether VA from food, after digestion and absorption, can be metabolized into RA in the body of Daurian ground squirrel and affect the gonads, this study divided male Daurian ground squirrels into three groups, which were fed with food containing different levels of VA throughout the year. Group 1VA was fed with food containing the standard amount of vitamin A (17 500 IU/kg), Group 2VA was fed with food containing twice the amount of vitamin A (35 000 IU/kg), and Group 0VA was fed with food without added vitamin A. At the end of hibernation, samples were taken to determine the morphological and histological indicators of the testis and epididymis, and to detect the differentiation of spermatogonia in the testes, as well as the levels of VA, RA, gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T) in the serum. The results showed that the levels of VA, RA, GnRH, LH, FSH, T in the serum, as well as the expression levels of retinoic acid gene 8 (Stra8) and meioticrecombination protein 8 (Rec8) in the testes of Spermophilus dauricus in Group 2VA and Group 1VA, were significantly higher than those in Group 0VA. However, there were no significant differences in the aforementioned indicators between Group 2VA and Group 1VA. This indicates that vitamin A can be metabolized into retinoic acid in the body of Daurian ground squirrel, promoting the development of gonads and spermatogenesis in male Daurian ground squirrels during seasonal reproduction. Adding an excessive amount of VA in the food has no significant effect on the gonads of male Daurian ground squirrels, but removing VA from the food does inhibit the differentiation of spermatogonia and the occurrence of sperm. This suggests that vitamin A in food is very important for the successful reproduction of Daurian ground squirrels under artificial breeding conditions.