OBJECTIVE To investigate the effect of Danhong Tongjing Prescription (DTP) on sperm quality in patients with bilateral varicocele (VC) after microsurgical varicocelectomy. METHODS We randomly assigned 68 patients with bilateral VC to receive microsurgical varicocelectomy (the control group, n = 34) or microsurgical varicocelectomy followed by oral administration of DTP for a course of 90 days (the DTP group, n = 34). Before and after treatment, we obtained the sperm concentration, total sperm count, total sperm motility, the percentage of progressively motile sperm (PMS), sperm acrosomal enzyme activity, inhibin B (Inh-B) level, and sperm DNA fragmentation index (DFI) from the patients and compared the parameters between the two groups. RESULTS There were no statistically significant differences in sperm concentration, PMS, acrosomal enzyme activity or sperm DFI among the patients with different degrees of VC preoperatively. After 3 months of medication, sperm concentration, total sperm count, total sperm motility, PMS and acrosomal enzyme activity were all increased while DFI decreased in both the control and DTP groups, even more significantly in the DTP group than in the control, and the Inh-B level was also markedly elevated in the DTP group in comparison with the baseline. CONCLUSIONS The severity of bilateral VC is not correlated with the reduction of semen quality. DTP can improve sperm quality by improving total sperm count, PMS and acrosomal enzyme activity and reducing DFI in VC patients after varicocelectomy. The underlying mechanisms of the prescription may be related to its anti-oxidative stress action and abilities of improving reproductive hypoxia, spermatogenic environment and the function of Sertoli cells, but the specific signaling pathway involved is not yet clear.