The transcription factor interferon regulatory factor 6 (IRF6) regulates craniofacial development and epidermal proliferation. We recently showed that IRF6 is a component of a regulatory feedback loop that controls the proliferative potential of epidermal cells. IRF6 is transcriptionally activated by p63 and induces its proteasome-mediated down-regulation, thereby limiting keratinocyte proliferative potential. We hypothesized that IRF6 may also be involved in skin carcinogenesis. Hence, we analyzed IRF6 expression in a large series of squamous cell carcinomas (SCCs) and found a strong down-regulation of IRF6 that correlated with tumor invasive and differentiation status. IRF6 down-regulation in SCC cell lines and primary tumors correlates with methylation on a CpG dinucleotide island located in its promoter region. To identify the molecular mechanisms regulating IRF6 potential tumor suppressive activity, we performed a genome-wide analysis by combining ChIP sequencing for IRF6 binding sites and gene expression profiling in primary human keratinocytes after siRNA-mediated IRF6 depletion. We observed dysregulation of cell cycle-related genes and genes involved in differentiation, cell adhesion, and cell-cell contact. Many of these genes were direct IRF6 targets. We also performed in vitro invasion assays showing that IRF6 down-regulation promotes invasive behavior and that reintroduction of IRF6 into SCC cells strongly inhibits cell growth. These results indicate a function for IRF6 in suppression of tumorigenesis in stratified epithelia.