The Wnt signaling pathway is a powerful and prominent oncogenic mechanism dysregulated in numerous cancer types. While evidence from transgenic mouse models and studies of human tumors clearly indicate that this pathway is of likely importance in human breast cancer, few clues as to the exact molecular nature of Wnt dysregulation have been uncovered in this tumor type. Here, we show that the Wnt inhibitory factor-1 (WIF1) gene, which encodes a secreted protein antagonistic to Wnt-dependent signaling, is targeted for epigenetic silencing in human breast cancer. We show that cultured human breast tumor cell lines display absent or low levels of WIF1 expression that are increased when cells are cultured with the DNA demethylating agent 5-aza-2'-deoxycytidine. Furthermore, the WIF1 promoter is aberrantly hypermethylated in these cells as judged by both methylation-specific PCR and bisulfite genomic sequencing. Using a panel of patient-matched breast tumors and normal breast tissue, we show that WIF1 expression is commonly diminished in breast tumors when compared with normal tissue and that this correlates with WIF1 promoter hypermethylation. Analysis of a panel of 24 primary breast tumors determined that the WIF1 promoter is aberrantly methylated in 67% of these tumors, indicating that epigenetic silencing of this gene is a frequent event in human breast cancer. Using an isogenic panel of cell lines proficient or deficient in the DNA methyltransferases (DNMTs) DNMT1 and/or DNMT3B, we show that hypermethylation of the WIF1 promoter is attributable to the cooperative activity of both DNMT1 and DNMT3B. Our findings establish the WIF1 gene as a target for epigenetic silencing in breast cancer and provide a mechanistic link between the dysregulation of Wnt signaling and breast tumorigenesis.