Aberrant activation of the Wingless-type (Wnt) signaling pathway is associated with a variety of human cancers, and we recently reported the importance of aberrant Wnt signaling in lung cancer. On the other hand, inhibition of Wnt signaling suppresses growth in numerous cell types. Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that can bind Wnt in the extracellular space and inhibit Wnt signaling. Recently, down-regulation of WIF-1 has been reported in several human cancers. To discover the mechanism of WIF-1 silencing in lung cancer, we first identified the human WIF-1 promoter and subsequently examined the methylation status in the CpG islands. By using methylation-specific PCR and sequence analysis after bisulfite treatment, we demonstrate here frequent CpG island hypermethylation in the functional WIF-1 promoter region. This hypermethylation correlates with its transcriptional silencing in human lung cancer cell lines. Moreover, treatment with 5-aza-2′-deoxycytidine restores WIF-1 expression. We then studied WIF-1 expression in 18 freshly resected lung cancers, and we show a down-regulation in 15 of them (83%). This silencing also correlates with WIF-1 promoter methylation. Our results suggest that methylation silencing of WIF-1 is a common and likely important mechanism of aberrant activation of the Wnt signaling pathway in lung cancer pathogenesis, raising its therapeutic interest.