This study aimed to investigate the impact of letrozole and crocin on Leydig cells on busulfan-induced azoospermia using a rat model. A sample population consisting of thirty male rats was randomly assigned to five groups: (1) the control group, (2) azoospermia group, (3) azoospermia group treated with letrozole, (4) azoospermia group treated with crocin, and (5) azoospermia group treated with both letrozole and crocin. The treatment period with letrozole and crocin lasted for four weeks following busulfan administration. Subsequently, comprehensive analyses, including histopathological, molecular, and hormonal assessments, were performed, followed by immunohistochemical staining. This study found that the control group exhibited normal Leydig cell morphology, while the azoospermia group showed reduced Leydig cells and tissue disruptions. Letrozole and crocin treatments were associated with increased testicular fibrosis in the AZO and AZO + Cro groups, while their combination notably reduced fibrosis to approximately 15%. Furthermore, the combination treatment enhanced antioxidant enzyme activity and upregulated androgen receptor expression. Although a number of improvements were noted in sperm motility, they were not statistically significant. Further research is required to clarify the therapeutic implications of these findings in azoospermia treatment.