Background: HER2 immunohistochemistry (IHC) is not routinely assessed in patients (pts) with aUC, but it is an emerging predictive biomarker with the advent of HER2-targeting agents. aUC outcomes with respect to HER2 status following treatment with immune checkpoint inhibitors (ICIs) and enfortumab vedotin (EV) are unknown. Methods: We retrospectively identified pts with aUC and available biopsies tested for HER2 IHC and fluorescence in situ hybridization (FISH). HER2 status was assessed using modified GI criteria as HER2 high (IHC 3+ or IHC 2+/FISH+), HER2 low (IHC 2+/FISH- or IHC 1+) or HER2 negative (IHC 0). Pt characteristics and outcomes were abstracted from chart review. We compared outcomes following ICI monotherapy and EV-based regimens in pts with HER2-high or HER2-low tumors relative to HER2-negative, and HER2-positive (≥IHC 1+) tumors relative to HER2-negative. Observed response rate (ORR) evaluated by local investigator was compared in pts with scans after ≥1 treatment cycles using logistic regression, while progression-free survival (PFS) and overall survival (OS) from treatment start were assessed using the Kaplan-Meier method and Cox proportional hazards model. Results: Biopsies from 181 pts with aUC obtained from 3/2016 – 3/2023 were tested for HER2 (34 high, 88 low, 58 negative, 1 indeterminate). In this group, 43 pts received ICI [38 (88%) pembrolizumab; 5 (12%) atezolizumab] and 37 EV [31 (82%) monotherapy; 6 (18%) combination regimen]. Pt characteristics and outcomes are shown in the Table. Among pts treated with EV, HER2-negative pts had decreased PFS (HR: 0.18, 95% CI 0.03 – 0.94, p=0.04) relative to HER2-high. No other differences were noted for any cross-group comparison. For pts treated with ICI, no differences in outcomes were observed for any comparisons based on HER2 status. Conclusions: In this single institution retrospective analysis, pts with aUC and HER2-high IHC expression had longer PFS relative to pts with HER2-negative expression when treated with EV-based regimens. No differences were observed in ICI outcomes based on HER2 expression. These hypothesis-generating results should be validated in larger cohorts.