1025 Background: DESTINY-Breast03 (NCT03529110), a randomized, multicenter, open-label, phase 3 study, assessed the efficacy and safety of T-DXd vs T-DM1 in pts with HER2+ mBC treated with ≥1 prior anti-HER2 regimen. Median (m) overall survival (OS) was not reached in either arm at the prior data cutoff (DCO; Jul 25, 2022). We report updated survival results, including efficacy and safety, after median duration of follow-up of 41 mo. Methods: Pts were randomized 1:1 to T-DXd 5.4 mg/kg or T-DM1 3.6 mg/kg once every 3 weeks. Primary endpoint was progression-free survival (PFS) by blinded independent central review (assessed at prior DCO); key secondary endpoint was OS. Other secondary endpoints included objective response rate (ORR), PFS, duration of response (DoR), PFS from time of randomization to progression on next line of therapy or death (PFS2), and safety. All secondary endpoints were based on investigator assessment. Results: 524 pts were randomized (T-DXd, n = 261; T-DM1, n = 263). As of Nov 20, 2023, median duration of follow-up was 43.0 mo (range, 0.0-62.9) for T-DXd and 35.4 mo (range, 0.0-60.9) for T-DM1. mOS (95% CI) was 52.6 mo (48.7-not evaluable [NE]) for T-DXd and 42.7 mo (35.4-NE) for T-DM1 (hazard ratio [HR], 0.73), with 110 (42.1%) and 126 (47.9%) OS events, respectively. OS rate (95% CI) at 36 mo was 67.6% (61.3-73.0%; T-DXd) vs 55.7% (49.2-61.7%; T-DM1). mPFS was 29.0 mo for T-DXd and 7.2 mo for T-DM1; PFS rate (95% CI) at 24 mo was 55.8% (49.1-62.0%; T-DXd) vs 20.6% (15.4-26.4%; T-DM1). mPFS2 was 45.2 mo (T-DXd) vs 23.1 mo (T-DM1). Median treatment duration was 18.2 mo (range, 0.7-56.6) with T-DXd vs 6.9 mo (range, 0.7-55.2) with T-DM1; rates of treatment-emergent adverse events (TEAEs) were consistent with prior DCO. Adjudicated drug-related interstitial lung disease/pneumonitis occurred in 16.7% of pts with T-DXd (4 new events [all grade 2] since prior DCO) vs 3.4% with T-DM1 (1 new event [grade 1]); neither arm had grade 4 or 5 events. Key efficacy and safety results are shown in the table. Conclusions: The superiority of T-DXd over T-DM1 was reinforced in this long-term analysis, as observed by clinically meaningful improvement in OS, PFS, and PFS2, consistent with prior DCO. The safety profile of T-DXd continues to be manageable with no cumulative toxicities observed with longer follow-up. Clinical trial information: NCT03529110 . [Table: see text]