Background: Long-term clinical benefit has been demonstrated in people with hemophilia A following a single administration of the investigational gene therapy valoctocogene roxaparvovec (AAV5-hFVIII-SQ). Safety, clinical effectiveness, and mechanisms of episomal vector DNA persistence have been previously described, but outstanding questions pertain to the maintenance of these attributes over increasing durations of follow-up. Aims: The four-year safety, efficacy, and durability of valoctocogene roxaparvovec is evaluated in a Phase 1/2 clinical study for severe hemophilia A. Methods: Adult male study participants with severe hemophilia A were followed for up to four years after receiving a single intravenous dose of valoctocogene roxaparvovec at 6×1013 vg/kg (n=7) or 4×1013 vg/kg (n=6). Results: After four (6×1013 vg/kg) or three (4×1013 vg/kg) years, all study participants demonstrated clinically meaningful FVIII activity levels with reductions in bleeds and FVIII usage. Following withdrawal from prophylaxis, annualized bleeding rate declined from pre-treatment mean by 95% at year four in 6×1013 vg/kg participants, and 93% at year three in 4×1013 vg/kg participants. Despite FVIII activity levels continuing to decline at a shallow rate, all patients in both cohorts remained off prophylaxis. After four years, the safety profile of valoctocogene roxaparvovec remained favorable and unchanged, with no inhibitor development or treatment-related ALT elevations beyond year one. Conclusions: Four-year follow-up data demonstrate that gene transfer with valoctocogene roxaparvovec leads to substantial and sustained FVIII activity levels, clinically relevant reductions in self-reported bleeding episodes, and significant reductions in FVIII replacement infusions. These data from the first-in-human trial represent the most up-to-date, long term follow-up data currently available for the investigational use of AAV-mediated therapy for hemophilia A.