N-acetyltransferase 2 (NAT2) is phase II enzyme with major roles in catalyzing the detoxification of aromatic amines, which are known risk factors for bladder cancer, and are ubiquitously present in the environment. We assessed the association between common polymorphisms in NAT2 gene and the risk of bladder cancer in 90 Slovak patients and 274 ethnicity-matched healthy controls. Effect modifications by smoking, age and gender were also evaluated. Overall, NAT2 slow acetylation was associated with significantly increased risk of bladder cancer (OR = 1.90; 95% CI, 1.15-3.16). In stratified analyses by age and gender, the elevated risk conferred by slow acetylator genotype was evident in older individuals (OR = 3.55; 95% CI, 1.77-7.35) and males (OR = 4.65; 95% CI, 1.68-16.10), with further increasing in NAT2*5B/*6A genotype carriers. Smoking was confirmed to be important risk factor, moreover, the risk was markedly increased in smokers with NAT2 slow acetylator genotype, and NAT2*5B/*6A carriers especially. In summary, these findings are consistent with previous literature suggesting that individual susceptibility to bladder cancer may be modulated by NAT2 polymorphisms, particularly in interaction with relevant environmental exposures such as smoking.