HER2-positive breast cancer patients carrying the germlineTSC2nonsynonymous variant c.4349 C > G (p.Pro1450Arg) are resistant to anti-HER2 therapy. Multi-predictor in silico analysis reveals that this variant is deleterious. We explore the potential mechanism of thisTSC2variant and investigate methods for overcoming anti-HER2 resistance.TSC2c.4349 C > G reverses the inhibitory effect on mTOR and downstream signaling by increasing TSC2 phosphorylation at Thr1462 and confers significant lapatinib resistance in vitro and in vivo. The combination of lapatinib and the CDK4/6 inhibitor palbociclib inhibits cyclin D1/CDK4/Rb alternative pathway and TSC2 phosphorylation, thereby partially attenuating mTOR activity and inducingTSC2-mutant cell blockage at G1/G0. In in vitro and xenograft models, palbociclib+lapatinib shows higher anti-tumor activity than monotherapy and overcomes the resistance of theTSC2c.4349 C > G-related variant to anti-HER2 therapy. We reveal a new mechanism of resistance to anti-HER2 therapy and provide a strategy to increase the efficiency of anti-HER2 therapy in HER2-positive breast cancer.