Abstract Uterine serous carcinoma (USC) is a rare, biologically aggressive variant of endometrial cancer with a high recurrence rate and poor prognosis. HER2 overexpression (3+ positivity) by IHC and/or FISH ERBB2 gene amplification is detected in approximately one-third of patients with USC. Clinical trials incorporating trastuzumab with standard chemotherapy have recently demonstrated improved progression-free and overall survival in advanced-stage or recurrent USC that overexpresses HER2. However, a large number of patients with USC eventually developed resistance to trastuzumab. Trastuzumab deruxtecan (T-DXd) is a novel HER2-directed antibody–drug conjugate with a topoisomerase I inhibitor payload recently approved by the Food and Drug Administration (FDA) for multiple tumor indications. Here, we investigated the in vitro and in vivo efficacy of T-DXd in primary USC cell lines and xenografts with different HER2 expression. T-DXd–induced cell growth suppression in HER2-overexpressing cell lines in vitro, increased early and late apoptosis as assessed by annexin and propidium iodide staining, and, similarly to trastuzumab, T-DXd–induced significant antibody-dependent cellular cytotoxicity in the presence of peripheral blood lymphocytes. While negligible activity was detected against USC cell lines with low HER2 expression, T-DXd demonstrated significant bystander killing against USC tumors with low/negligible HER2 when such cells were admixed with HER2 3+ tumor cells in vitro. T-DXd showed tumor growth suppression in in vivo USC PDX models that overexpress HER2 at 3+ levels, prolonging survival when compared with controls, with minimal toxicity. Future clinical trials are warranted in patients with USC failing trastuzumab treatment.