Abstract Nitric oxide signaling plays complex roles in carcinogenesis, in part, due to incomplete mechanistic understanding. In this study, we investigated our discovery of an inverse correlation in the expression of the inducible nitric oxide synthase (iNOS) and the Wnt/β-catenin regulator Dickkopf-1 (DKK1) in human cancer. In human tumors and animal models, induced nitric oxide synthesis increased Wnt/β-catenin signaling by negatively regulating DKK1 gene expression. Human iNOS (hiNOS) and DKK1 gene expression were inversely correlated in primary human colon and breast cancers, and in intestinal adenomas from Min (Apcmin/+) mice. Nitric oxide production by various routes was sufficient to decrease constitutive DKK1 expression, increasing Wnt/β-catenin signaling in colon and breast cancer cells and primary human hepatocytes, thereby activating the transcription of Wnt target genes. This effect could be reversed by RNA interference-mediated silencing of iNOS or treatment with iNOS inhibitors, which restored DKK1 expression and its inhibitory effect on Wnt signaling. Taken together, our results identify a previously unrecognized mechanism through which the nitric oxide pathway promotes cancer by unleashing Wnt/β-catenin signaling. These findings further the evidence that nitric oxide promotes human cancer and deepens insights in the complex control Wnt/β-catenin signaling during carcinogenesis. Cancer Res; 73(21); 6526–37. ©2013 AACR.