Varicocele is one of the most common causes of primary male infertility. Pilea microphylla (PM) is being used as folk medicine. This study was aimed to investigate the effects of PM in a rat model of varicocele. A total of 30 male Wistar rats were divided into control, sham, varicocele, accessory varicocele and PM-treated groups. After 10 weeks of varicocele induction, sperm parameters and chromatin (Aniline blue, acridine orange and toluidine blue) were evaluated, except for the treated and accessory groups that received 50 mg/kg PM orally daily for 10 weeks and then were sacrificed. Sperm parameters significantly decreased in varicocele groups (P < 0.01). Moreover, there was a negative correlation between the DNA fragmentation and sperm parameters in varicocelized rats. Administration of PM led to significantly increased sperm parameters and AO staining (P < 0.05). These findings suggest that PM improves sperm parameters and DNA fragmentation in varicocelized rats. PM can reduce the damage to sperm DNA but not chromatin condensation.