Tuberous sclerosis complex (TSC) is a genetic disease caused by mutations in either TSC1 or TSC2 tumor suppressor genes. TSC1 and TSC2 (also known as hamartin and tuberin, respectively) form a functional complex and negatively regulate cell growth by inhibiting protein synthesis. 14-3-3 binds to TSC2 and may inhibit TSC2 function. We have reported previously that phosphorylation of serine 1210 (Ser(1210)) in TSC2 is essential for 14-3-3 binding. Here we show that serum and anisomycin enhance the interaction between TSC2 and 14-3-3 by stimulating phosphorylation of Ser(1210). Activation of p38 MAP kinase (p38) is essential for the stimulating effect of serum and anisomycin although p38 is not directly responsible for the phosphorylation of Ser(1210) in TSC2. Both in vitro and in vivo experiments demonstrate that the p38-activated kinase MK2 (also known as MAPKAPK2) is directly responsible for the phosphorylation of Ser(1210). Our data show that anisomycin stimulates phosphorylation of Ser(1210) of TSC2 via the p38-MK2 kinase cascade. Phosphorylation of TSC2 by MK2 creates a 14-3-3 binding site and thus regulates the cellular function of the TSC2 tumor suppressor protein.