MAPK cascades play the critical role in regulating Ras oncogene activity by phosphorylation-dependent mechanisms. Whereas the ERK MAPK pathway is required for Ras transformation, our previous works established that the p38 activity is inhibitory to Ras signaling in both experimental and ras-mutated cancer cells [Chen, G., Hitomi, M., Han, J., and Stacey, D. W. (2000) J. Biol. Chem. 275, 38973–38980; Qi, X., Tang, J., Pramanik, R., Schultz, R. M., Shirasawa, S., Sasazuki, T., Han, J., and Chen, G. (2004) J. Biol. Chem., 279, 22138–22144]. Here we report that K-Ras activates p38γ, a p38 MAPK family member, by inducing its expression without increasing its phosphorylation and depletion of induced p38γ suppresses Ras transformation in rat intestinal epithelial cells. This p38γ activity contrasts with that of its family member p38α, which is activated by Ras through phosphorylation, leading to an inhibition of Ras transformation. Mechanistic analyses show that unphosphorylated p38γ may promote Ras transformation through an increased complex formation with ERK proteins. Significantly, functional p38γ protein is expressed only in K-ras mutated human colon cancer cells, and p38γ transcripts are ubiquitously increased in a set of primary human colon cancer tissues. These studies thus demonstrate the essential role of p38γ in K-Ras transformation independent of phosphorylation and elevated p38γ may serve as a novel diagnostic marker and therapeutic target for human colon cancer.