Peroxisome proliferator-activated receptor gamma (PPARgamma) causes epithelial to mesenchymal transformation (EMT) in intestinal epithelial cells, as evidenced by reorganization of the actin cytoskeleton, acquisition of a polarized, mesenchymal cellular morphology, increased cellular motility, and colony scattering. This response is due to activation of Cdc42, resulting in p21-activated kinase-dependent phosphorylation and activation of MEK1 Ser(298) and activation of ERK1/2. Dominant negative MEK1, MEK2, and ERK2 block PPARgamma-induced EMT, whereas constitutively active MEK1 and MEK2 induce a mesenchymal phenotype similar to that evoked by PPARgamma. PPARgamma also stimulates ERK1/2 phosphorylation in the intestinal epithelium in vivo. PPARgamma induces the p110alpha subunit of phosphoinositide 3-kinase (PI3K), and inhibition of PI3K blocks PPARgamma-dependent phosphorylation of MEK1 Ser(298), activation of ERK1/2, and EMT. We conclude that PPARgamma regulates the motility of intestinal epithelial cells through a mitogen-activated protein kinase cascade that involves PI3K, Cdc42, p21-activated kinase, MEK1, and ERK1/2. Regulation of cellular motility through Rho family GTPases has not been previously reported for nuclear receptors, and elucidation of the mechanism that accounts for the role of PPARgamma in regulating motility of intestinal epithelial cells provides fundamental new insight into the function of this receptor during renewal of the intestinal epithelium.