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Background: NAT2 enzyme involved in bioconversion of aromatic amines, heterocyclic arylamines and certain drugs into electrophilic ions that can be important initiators in tumorigenesis process. Objectives: The aim of this study was to assess the possible association between NAT2 polymorphisms (857 G > A, 481 T > C, and 590 G > A) and risk of prostate cancer (PC). Methods: Totally, 207 benign prostate hyperplasia (BPH) and 147 PC Iranian patients were evaluated. NAT2 genotypes were detected by restriction fragment length polymorphism (RFLP). Multiple logistic regression models were used to estimate the odds ratios for the association between presence of each genotype and developing PC. Results: For NAT2 G857A, the frequency of AA and AG genotypes was lower among PC patients compared to those without it (1.01% vs. 0 and 55.88% vs. 54.55%, respectively; P = 0.7). For NAT2 T481c, the odds ratios for the association of TT and CT genotypes with PC were 0.65 and 0.55, respectively, which were not statistically significant (P = 0.5 and P = 0.09, respectively). For NAT2 G590a, both AA (11.11% vs. 12.87%) and AG (45.83% vs. 52.48%) genotypes were significantly more common among PC patients compared to BPC patients (P = 0.008). However, none of the relevant odds ratios were statistically significant (OR = 2.2, P = 0.2 and OR = 1.72, P = 0.1, respectively). Among PC patients, CT genotype of T481C caused more than 4-fold significant increase in the risk of developing advanced stages of PC. Conclusions: Our study represented credible evidence that carrying G857A, G590A and T481C polymorphisms of NAT2 may not affect developing PC, its grading or invasion, but heterozygote genotype of T481C polymorphism (Rapid acetylator) can be associated with more advanced stages of cancer earlier in life. Further longitudinal studies with larger sample sizes are needed to more precisely assess the genetic risk factors of PC.