Defective hepatic autophagy is observed in obesity and diabetes, whereas autophagy is inhibited by insulin in hepatocytes. Insulin-induced anti-autophagy is mediated by non-canonical Gαi3 signaling via an unknown mechanism. Previously, we identified the anti-autophagic activity of Tnfaip8 via activation of mammalian target of rapamycin (mTOR) in the nervous system. Here, we demonstrate that insulin temporally induces Tnfaip8, which mediates the anti-autophagic action of insulin through formation of a novel ternary complex including Tnfaip8, phosphatidylethanolamine (PE) and Gαi3. Specifically, an X-ray crystallographic study of Tnfaip8 from Mus musculus (mTnfaip8) at 2.03 Å together with LC-MS analyses reveals PE in the hydrophobic cavity. However, an mTnfaip8 mutant lacking PE does not interact with Gαi3, indicating that the PE component is critical for the anti-autophagic action of mTnfaip8 via interaction with Gαi3. Therefore, the mTnfaip8-PE complex may act as an essential upstream effector via ternary complex formation most likely with active Gαi3 during insulin-induced anti-autophagy.