ARX788 is a novel antibody-drug conjugate (ADC) that combines an anti-HER2 mAb with a potent tubulin inhibitor payload AS269 through site-specific conjugation to the antibody. AS269 is an unnatural amino acid. Results from a phase I research of ARX788’s safety, pharmacokinetics, and anticancer efficacy in patients with HER2-positive metastatic breast cancer (MBC) are presented here. Doses of 0.33, 0.66, 0.88, 1.1, 1.3, or 1.5 mg/kg of ARX788 were given every 3 weeks or every 4 weeks to patients with HER2-positive MBC. Dose-limiting toxicity (DLT) was evaluated over 84 days for pulmonary toxicity and over 1 cycle (21 or 28 days) for other toxicities. A total of 69 patients participated. There were no drug-induced or fatality-causing accidents. At least 1 treatment-related adverse event occurred in 97.1% of patients (67/69). Treatment-related adverse event (TRAE). Increases in aspartate aminotransferase and alanine aminotransferase, corneal epitheliopathy, alopecia, hypokalemia, interstitial lung disease (ILD)/pneumonitis, and aldosterone were among the most frequently reported adverse events (TRAEs; prevalence ≥30%). While ILD/pneumonia affected 34.8% of individuals, only 2% of those cases were severe. The objective response rate was 65.5% [19/29, 95% CI, 45.7-82.1] at the recommended phase II dose of 1.5 mg/kg every 3 weeks, the disease control rate was 100% (95% CI, 81.2-100), and the median progression-free survival was 17.02 months (95% CI, 10.09-not attained). Patients with HER2-positive MBC whose cancer had progressed on earlier anti-HER2 therapy showed a tolerable safety profile and early signs of efficacy with ARX788.