DNA variation inInterferon Regulatory Factor 6(IRF6) causes Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate (CLP). However, an etiologic variant inIRF6has been found in only 70% of VWS families. To test whether DNA variants in regulatory elements cause VWS, we sequenced three conserved elements nearIRF6in 70 VWS families that lack an etiologic mutation withinIRF6exons. A rare mutation (350dupA) was found in a conservedIRF6enhancer element (MCS9.7) in a Brazilian family. The350dupAmutation abrogated the binding of p63 and E47 transcription factors to cis-overlapping motifs, and significantly disrupted enhancer activity in human cell cultures. Moreover, using a transgenic assay in mice, the350dupAmutation disrupted the activation ofMCS9.7enhancer element and led to failure oflacZexpression in all head and neck pharyngeal arches. Interestingly, disruption of the p63 Motif1 and/or E47 binding sites by nucleotide substitution did not fully recapitulate the effect of the350dupAmutation. Rather, we recognized that the350dupAcreated a CAAAGT motif, a binding site for Lef1 protein. We showed that Lef1 binds to the mutated site and that overexpression of Lef1/β-Catenin chimeric protein repressedMCS9.7-350dupAenhancer activity. In conclusion, our data strongly suggest that350dupAvariant is an etiologic mutation in VWS patients and disrupts enhancer activity by a loss- and gain-of-function mechanism, and thus support the rationale for additional screening for regulatory mutations in patients with CLP.