<p>The Rab GTPase activating protein (RabGAP) AS160 translocates from the cytosol into the nucleus acting as a transcriptional co-activator of Signal Transducer and Activator of Transcription 3 (STAT3) to regulate proliferation of muscle satellite cells (MuSCs). How this AS160–STAT3 complex is regulated remains largely unclear yet. Here, we show that TBC1D1, a RabGAP related to AS160, forms a super-complex with AS160 and STAT3 to retain the AS160–STAT3 complex in the cytosol. Phosphorylation of TBC1D1-Thr⁵⁹⁶ by protein kinase B dissociates TBC1D1 from AS160 thus releasing the cytosolic retention of the AS160–STAT3 complex. A non-phosphorylatable alanine substitution of Thr⁵⁹⁶ inhibits MuSC proliferation and impairs repair of injured muscle. In contrast, TBC1D1 deficiency, but not its GAP-inactive mutation, promotes MuSC proliferation and muscle regeneration. Thus, TBC1D1 is a negative regulator of MuSC proliferation through cytosolic retention of the AS160–STAT3 complex and might be a valuable therapeutic target for muscle regeneration.</p>