Phosphoinositide 3-kinase (PI(3)K) is a unique enzyme characterized by both lipid and protein kinase activities. Here, we demonstrate a requirement for the protein kinase activity of PI(3)K in agonist-dependent β-adrenergic receptor (βAR) internalization. Using PI(3)K mutants with either protein or lipid phosphorylation activity, we identify the cytoskeletal protein non-muscle tropomyosin as a substrate of PI(3)K, which is phosphorylated in a wortmannin-sensitive manner on residue Ser 61. A constitutively dephosphorylated (S61A) tropomyosin mutant blocks agonist-dependent βAR internalization, whereas a tropomyosin mutant that mimics constitutive phosphorylation (S61D) compliments the PI(3)K mutant, with only lipid phosphorylation activity reversing the defective βAR internalization. Notably, knocking down endogenous tropomyosin expression using siRNAs that target different regions of tropomyosin resulted in complete inhibition of βAR endocytosis, showing that non-muscle tropomyosin is essential for agonist-mediated receptor internalization. These studies demonstrate a previously unknown role for the protein phosphorylation activity of PI(3)K in βAR internalization and identify non-muscle tropomyosin as a cellular substrate for protein kinase activity of PI(3)K.