HER2 mutations drive the growth of a subset of breast cancers and are targeted with HER2 tyrosine kinase inhibitors (TKI) such as neratinib. However, acquired resistance is common and limits the durability of clinical responses. Most HER2-mutant breast cancers progressing on neratinib-based therapy acquire secondary mutations in HER2. It is unknown whether these secondary HER2 mutations, other than the HER2T798I gatekeeper mutation, are causal to neratinib resistance. Herein, we show that secondary acquired HER2T862A and HER2L755S mutations promote resistance to HER2 TKIs via enhanced HER2 activation and impaired ner-atinib binding. While cells expressing each acquired HER2 mutation alone were sensitive to neratinib, expression of acquired double mutations enhanced HER2 signaling and reduced neratinib sensi-tivity. Computational structural modeling suggested that secondary HER2 mutations stabilize the HER2 active state and reduce ner-atinib binding affinity. Cells expressing double HER2 mutations exhibited resistance to most HER2 TKIs but retained sensitivity to mobocertinib and poziotinib. Double-mutant cells showed enhanced MEK/ERK signaling, which was blocked by combined inhibition of HER2 and MEK. Together, these findings reveal the driver function of secondary HER2 mutations in resistance to HER2 inhibition and provide a potential treatment strategy to overcome acquired resistance to HER2 TKIs in HER2-mutant breast cancer.Significance: HER2-mutant breast cancers acquire secondary HER2 mutations that drive resistance to HER2 tyrosine kinase inhibitors, which can be overcome by combined inhibition of HER2 and MEK.