The type II transforming growth factor‐β (TGF‐β) receptor Ser/Thr kinase (TβRII) is responsible for the initiation of multiple TGF‐β signaling pathways, and loss of its function is associated with many types of human cancer. Here we show that TβRII kinase is regulated intricately by autophosphorylation on at least three serine residues. Ser213, in the membrane‐proximal segment outside the kinase domain, undergoes intra‐molecular autophosphorylation which is essential for the activation of TβRII kinase activity, activation of TβRI and TGF‐β‐induced growth inhibition. In contrast, phosphorylation of Ser409 and Ser416, located in a segment corresponding to the substrate recognition T‐loop region in a three‐dimensional structural model of protein kinases, is enhanced by receptor dimerization and can occur via an inter‐molecular mechanism. Phosphorylation of Ser409 is essential for TβRII kinase signaling, while phosphorylation of Ser416 inhibits receptor function. Mutation of Ser416 to alanine results in a hyperactive receptor that is better able than wild‐type to induce TβRI activation and subsequent cell cycle arrest. Since on a single receptor either Ser409 or Ser416, but not both simultaneously, can become autophosphorylated, our results show that TβRII phosphorylation is regulated intricately and affects TGF‐β receptor signal transduction both positively and negatively.