RGS proteins negatively regulate heterotrimeric G protein signaling. Recent reports have shown that RGS proteins modulate neuronal, cardiovascular, and lymphocytic activity, yet their role in carcinogenesis has not been explored. In an epidemiologic study of 477 bladder cancer patients and 446 matched controls, three noncoding single-nucleotide polymorphisms (SNPs) in RGS2 and RGS6 were each associated with a statistically significant reduction in bladder cancer risk. The risk of bladder cancer was reduced by 74% in those individuals with the variant genotype at all three SNPs (odds ratio, 0.26; 95% confidence interval, 0.09-0.71). When the SNPs were analyzed separately, the RGS6-rs2074647 (C-->T) polymorphism conferred the greatest overall reduction in risk of bladder cancer (odds ratio, 0.66; 95% confidence interval, 0.46-0.95). These reductions in risk were more pronounced in ever smokers, suggesting a gene-environment interaction. In transfection assays, the RGS6-rs2074647 (C-->T) polymorphism increased the activity of a luciferase-RGS fusion protein by 2.9-fold, suggesting that this SNP is functionally significant. Finally, we demonstrate that RGS2 transcripts and several splice variants of RGS6 are expressed in bladder cancer cells. These data provide the first evidence that RGS proteins may be important modulators of cancer risk and validate RGS6 as a target for further study.