Objective: In this study, the effects of Lactobacillus acidophilus on testosterone (TES), follicle-stimulating hormone (FSH), luteinizing hormone (LH), androgen-binding protein (ABP), factor-associated apoptosis (FAS), and total cholesterol (TC), as well as histopathological changes, were investigated in male rats fed a high-cholesterol diet. Methods: The study included three groups. The control (C) group was fed standard-diet for 8 weeks. The hypercholesterolemia (HC) group was fed a 2% cholesterol-diet for 8 weeks. The therapeutic group (HCL) was fed a 2% cholesterol-diet for 8 weeks and administered L. acidophilus for the last 4 weeks. FSH, TES, and FAS levels in testicular tissue were determined using an enzyme-linked immunosorbent assay (ELISA), while another sample was examined histopathologically. LH and ABP levels were determined using ELISA, and serum TC levels were assessed via an autoanalyzer. Results: In the HC group, the TC levels were significantly higher and the LH levels were lower (p<0.05) than in the C group. The ABP levels were lower (p>0.05). In the HCL group, the LH and ABP levels were higher (p>0.05) and the TC level significantly lower (p<0.05) than in the HC group. The TES and FSH levels were lower, and the FAS levels were higher, in the HC than in the C group (p<0.05). In the HCL group, levels of all three resembled control levels. Histologically, in the testicular tissue of the HC group, the cells in the tubular wall exhibited atrophy, vacuolization, and reduced wall structure integrity. However, in the HCL group, these deteriorations were largely reversed. Conclusion: Supplementary dietary administration of an L. acidophilus to hypercholesterolemic male rats positively impacted testicular tissue and male fertility hormone levels.