The prescription “Huang's SiYu Pill” (HSYP) was founded by professor Huang Haibo, a famous traditional Chinese medicine in Inner Mongolia, based on long-term clinical experience. In this study we observed the effects of HYSP on sperm quality, hormone level and the expression level of reproductive related genes and proteins in testicular stromal cells of rats with kidney-yin deficiency to explore the therapeutic mechanism of HSYP in the treatment of infertility with kidney-yin deficiency, Liuwei Dihuang Pill group (LDP) and Tamoxifen Citrate Tablets combined with Pancreatic Kallidinogenase Enteric Coated Tablets were used to be the positive control drug. After 25 days of medicine administration, the animals were anesthetized and killed, the serum and testicular tissues were retained for further experiments. The ELISA results showed that HSYP had a better effect on increasing serum FSH level than the conventional Chinese medicine or western medicine, while there was no remarkable difference in the regulation of serum LH in each administration group. The results of RT-qPCR showed that the transcription levels of Cyp11a1, Hsd3b1, Lhcgr and Star genes in the medium and high dose groups of HSYP were significantly upregulated compared with the model group of kidney-yin deficiency, while the expression of Nr5a1 gene was not significantly affected. The results of HE and immunohistochemistry showed that HSYP significantly improved the development level of testicular stromal cells and promoted the expression levels of CYP11A1 and HSD11B1 in testicular stromal cells, compared with the model group of kidney-yin deficiency. Together, our results showed that Huang's SiYu Pills can significantly improve the development and function of Leydig cells in SD rats with kidney-yin deficiency model, which provided a good research basis for deep research on relevant pharmacological mechanisms of the medicine.