Activating mutations in HER2 (ERBB2) drive the growth of a subset of breast and other cancers and tend to co-occur with HER3 (ERBB3) missense mutations. The HER2 tyrosine kinase inhibitor neratinib has shown clinical activity against HER2-mutant tumors. To characterize the role of HER3 mutations in HER2-mutant tumors, we integrate computational structural modeling with biochemical and cell biological analyses. Computational modeling predicts that the frequent HER3E928G kinase domain mutation enhances the affinity of HER2/HER3 and reduces binding of HER2 to its inhibitor neratinib. Co-expression of mutant HER2/HER3 enhances HER2/HER3 co-immunoprecipitation and ligand-independent activation of HER2/HER3 and PI3K/AKT, resulting in enhanced growth, invasiveness, and resistance to HER2-targeted therapies, which can be reversed by combined treatment with PI3Kα inhibitors. Our results provide a mechanistic rationale for the evolutionary selection of co-occurring HER2/HER3 mutations and the recent clinical observations that HER3 mutations are associated with a poor response to neratinib in HER2-mutant cancers. Display omitted •Co-occurring HER2/HER3 mutations promote oncogenesis and invasion via PI3K activation•HER3 mutations reduce sensitivity to HER2 inhibitors in HER2-mutant cancer cells•Tumors with HER2/HER3 mutations are sensitive to HER2 TKI + PI3Kα inhibitor Hanker and Brown et al. demonstrate that co-occurring HER2 and HER3 mutations cooperatively activate HER2/HER3 and PI3K signaling in tumor cells, leading to enhanced growth, invasion, and resistance to HER2 inhibitors. HER2/HER3 double-mutant tumor models are sensitive to the combination of a HER2 TKI and a PI3Kα inhibitor.