Background: Varicocele is one of the common causes of male infertility, associated with increased oxidative stress and damage to sperm DNA. This study aims to investigate the effects of L-cysteine, taurine, and vitamin B6 (LTB) on spermatogenesis, sperm function, and enzymes involved in the trans-sulfuration pathway in the testes of varicocele-induced rats. Methods: Male Wistar rats (180–220 g) were randomly divided into five experimental groups: the control group (no intervention), the sham group (simple laparotomy), the varicocele group (V), the LTB-treated control group, and the LTB-treated varicocele group. Evaluations included sperm parameter assessments, testicular histology analysis, measurement of testosterone levels, vitamin B6 levels, various amino acids, and the expression of enzymes in the trans-sulfuration pathway at the protein level using the Western blot method. Results: The study revealed a significant reduction in sperm quality parameters and sperm function (p<0.05) in the varicocele group compared to the control and sham groups. Additionally, enzymes involved in the trans-sulfuration pathway were reduced in the varicocele group compared to the sham group, although the difference was not statistically significant. Plasma B6 levels in the varicocele group showed a significant decrease compared to the control group (p<0.05). Supplementation with LTB improved the negative effects of varicocele on spermatogenesis and sperm function. Conclusion: Antioxidants may play a role in improving varicocele. Using amino acids and vitamins involved in the one-carbon cycle and the trans-sulfuration pathway can enhance the testicular antioxidant system and counteract the negative effects of varicocele. These findings highlight the need for further studies and the potential for higher doses or longer durations of antioxidant supplementation to achieve better therapeutic effects.