Patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer eventually develop resistance to dual-antibody therapy with trastuzumab plus pertuzumab. Mechanisms of resistance have not been well elucidated. We evaluated the safety, tolerability, and efficacy of ado-trastuzumab emtansine (T-DM1) plus neratinib in patients who progressed on trastuzumab plus pertuzumab. In this 3 + 3 dose-escalation study, patients with metastatic breast cancer who progressed on trastuzumab, pertuzumab, and a taxane were treated with T-DM1 at 3.6 mg/kg intravenously every 3 weeks and dose-escalating neratinib at 120, 160, 200, or 240 mg/d orally. Twenty-seven patients were treated across four dose-levels of neratinib. Dose-limiting toxicity in cycle 1 was grade 3 diarrhea in six patients and grade 3 nausea in one; no patient experienced grade 4 diarrhea, and there were no grade 5 toxicities. Other grade 3 to 4 toxicities included nausea (11%), dehydration (11%), electrolyte abnormality (19%), thrombocytopenia (15%), elevated transaminase levels (7%), and fatigue (7%). Twelve (63%) of 19 evaluable patients had an objective response. Responses occurred at all neratinib doses. Plasma cell-free DNA at baseline showed (HER2) amplification in 10 of 27 patients. Deep and more durable responses occurred in patients with cell-free DNA amplification. Two complete responders had high expression of total HER2 and p95HER2 in baseline tissue. We report the recommended phase II dose of T-DM1 3.6 mg/kg and neratinib 160 mg/d for this combination. Possible resistance mechanisms to HER2 antibodies may be loss of the HER2 receptor and high expression of p95HER2. These data provide the basis for an ongoing phase II study to better define the activity of this regimen.