Kallikarein‐related peptidase 3 (KLK3) gene polymorphisms seem to play a role in susceptibility to prostate cancer (PC). The purpose of this study was to investigate the association between rs2735839 polymorphism of KLK3 gene and risk of PC in an Iranian population. In this case‐control study, rs2735839 was genotyped in 532 patients with PC and 602 controls with benign prostate hyperplasia (BPH) using polymerase chain reaction‐restriction fragment length polymorphism assay. The frequency of GG, AG, and AA genotypes of KLK3 polymorphism was 24.6% and 76.2%, 46.6% and 21.7%, and 28.8% and 2.1%, in patients with BPH and PC, respectively (P < 0.001). The frequency of G allele in patients with BPH and PC was 47.9% and 87%, respectively (odds ratio: 7.31; confidence interval: 5.88‐9.10; P < 0.001). Patients with AG and GG genotypes had a higher total serum level of prostate‐specific antigen (PSA) compared to those with AA genotype (P < 0.001). Patients with this polymorphism had higher risk of tumor with higher grade (P = 0.23), advanced stage (P = 0.11), perineural invasion (P = 0.07), and vascular invasion (P = 0.07) compared to those without it but this difference was not statistically significant. Based on our results, KLK3 gene polymorphism was associated with the risk of PC. Higher levels of PSA in the presence of KLK3 polymorphism in patients with PC indicated that rs2735839 polymorphism could be a risk factor for increased levels of PSA.