Objective: To investigate the cytotoxicity of Smilax myosotiflora (S. myosotiflora) methanolic extract and its effects on sexual hormone levels and testicular histology in male rats. Methods: The cytotoxicity of S. myosotiflora methanolic extract was investigated by employing brine shrimp lethality assay. Forty eight male rats were randomly divided into four groups (Groups I-IV) of 12 each. Rats in Group I were administered with 0.5 mL of distilled water (vehicle), whilst Groups II, III and IV received 200, 400 and 800 mg/kg of the methanolic extract of S. myosotiflora in 0.5 mL of the vehicle, respectively. Male rats treated with continuous daily dosing were killed and necropsied after a total dose period of 60 days. Sexual hormones were assayed and histological examination of testes was performed according to standard methods. Results: S. myosotiflora extracts did not produce any cytotoxicity to brine shrimp in all concentrations tested. Serum testosterone level was significantly higher in rats treated with high dose of S. myosotiflora. Testicular histology showed normal architecture with all stages of spermatogenesis in all experimental groups. Conclusions: The present work confirmed that S. myosotiflora extract improves reproductive functions, without any cytotoxic activity and produces no histological changes to the testes.