Activating mutations in the K-Ras small GTPase are extensively found in human tumors. Although these mutations induced the generation of a constitutively GTP-loaded, active form of K-Ras, phosphorylation at Ser181 within the C-terminal hypervariable region is able to modulate oncogenic K-Ras function without affecting the in vitro affinity for its effector Raf-1. In striking contrast, K-Ras phosphorylated at Ser181 showed an increased interaction with active form of Raf-1 and for PI3K (p110α) in cells. As most phosphorylated K-Ras is located at the plasma membrane, differential localization within this membrane according to the phosphorylation status was explored. Plasma membrane density gradient fractionation in the absence of detergents showed segregation of phosphomimetic and non-phosphorylatable K-Ras mutants (Ser181D and Ser181A, respectively). Moreover, immuno-electron-microscopy-statistics analysis showed that both phosphorylation mutants form distinct non-overlapping nanoclusters. Finally, promotion or inhibition of oncogenic K-Ras phosphorylation by PKC increased its co-clustering with the phosphomimetic or the non-phosphorylatable mutant, respectively. Most interestingly, PI3K (p110α) was found in phosphorylated and excluded in non-phosphorylated K-Ras nanoclusters. In conclusion, our data provide for the first time evidences that phosphorylation of oncogenic K-Ras by PKC induces segregation of K-Ras in spatially distinct nanoclusters at the plasma membrane which in turn would favor Raf-1 and PI3K activation.