Zishen Yutai Pill (ZYP), containing 15 Chinese traditional medicine, is a safe and well quality-controlled TCM preparation with promising effects in many fields of reproduction. The current study was designed to investigate the therapeutic effects of ZYP on sperm quality and testis in varicocele (VC) rats.Male Wistar rats were randomly divided into four groups (n = 6), i.e., a sham group, a VC group, and VC groups treated with different dose of ZYP (1575 and 3150 mg/kg/d, respectively). The experimental VC model was established by partial ligation of left renal vein. Six weeks after model establishment, ZYP was orally administered once a day for the next 6 weeks. Parameters relating to testis and sperm quality were assessed. Hematoxylin-eosin staining was used to showed testicular tissue damage in experimental VC rats. Expressions of proteins relating to NLRP3 inflammasome pathways were determined using Western blot (WB). The mRNA expressions of relating genes were determined using quantitative real-time PCR (qRT-PCR) analysis.ZYP could significantly improve sperm motility and decrease sperm DNA fragmentation index in VC rats (P < 0.05). Hematoxylin-eosin (HE) staining showed that ZYP could alleviate testicular tissue damage caused by experimental varicocele in rats. Compared to the VC model, expressions of NLRP3, ASC, and caspase-1 in rats treated with ZYP were significantly down-regulated, as validated by both qRT-PCR and WB analysis (P < 0.05).In brief, ZYP could improve sperm DNA integrity by inhibiting the NLRP3 inflammasome pathway and alleviating the chronic inflammation of testicular tissue induced by experimental varicocele in rats.© 2023 The Authors.