Fat, a candidate tumor suppressor in Drosophila, is a component of Hippo signaling pathway involved in controlling organ size. We found that a ∼3 Mbp deletion in mouse chromosome 3 caused tumorigenesis of a non‐tumorigenic mammary epithelial cell line. The expression of Fat4 gene, one member of the Fat family, in the deleted region was inactivated, which resulted from promoter methylation of another Fat4 allele following the deletion of one Fat4 allele. Re‐expression of Fat4 in Fat4‐deficient tumor cells suppressed the tumorigenecity whereas suppression of Fat4 expression in the non‐tumorigenic mammary epithelial cell line induced tumorigenesis. We also found that Fat4 expression was lost in a large fraction of human breast tumor cell lines and primary tumors. Loss of Fat4 expression in breast tumors was associated with human Fat4 promoter methylation. Together, these findings suggest that Fat4 is a strong candidate for a breast tumor suppressor gene. © 2008 Wiley‐Liss, Inc.