To investigate the mechanism by which Yishen Tongluo Formula-containing serum regulates ferroptosis of spermatocytes to treat varicocele(VC)-induced infertility.Methods:1) Metabolomics analysis was performed on normal and VC human spermatogenic vein endothelial cells cultured in vitro to identify significantly different pathogenic metabolites. 2) A mouse spermatocyte cell line was cultured，and the optimal concentration of Yishen Tongluo Formula-containing serum and pathogenic metabolites was determined using the CCK-8 assay.Cells were randomly divided into the normal group，model group(metabolite group)，and the formula-containing serum group(metabolite+Yishen Tongluo Formula group).Ferroptosis in each group was observed.The expression of ferroptosis-related proteins，including glutathione peroxidase 4(GSH-Px4)，solute carrier family 7 member 11(SLC7A11)，acyl-CoA synthetase long-chain family member 4(ACSL4)，and ferritin(Fn)，and their mRNA levels were measured by Western blot and real-time quantitative PCR.Results:1) The ferroptosis metabolic pathway was closely related to VC，and oxidized glutathione(GSSG) was identified as the main pathogenic metabolite. 2) Compared to the normal group，the cell viability in the model group significantly decreased(P<0.01)，and the expression of GSH-Px4，SLC7A11，and Fn proteins and mRNA was downregulated，while ACSL4 protein and mRNA expression was upregulated(P<0.05，P<0.01).Compared with the model group，the drug-treated groups showed a significant increase in cell viability(P<0.01)，upregulation of GSH-Px4，SLC7A11，and Fn proteins and mRNA expression，and downregulation of ACSL4 protein and mRNA expression(P<0.01).Conclusion:1) The ferroptosis metabolic pathway is closely related to VC-induced infertility，and GSSG is identified as a key pathogenic metabolite. 2) Yishen Tongluo Formula may exert its therapeutic effect on VC-induced infertility by inhibiting GSSG and modulating the expression of ferroptosis-related proteins in spermatocytes.