The Muruntau gold deposit, Uzbekistan, is one of the largest gold deposits known worldwide, but its origin remains controversial. We used Re-Os arsenopyrite geochronology to precisely determine the age of main-stage gold mineralization at Muruntau to be 287.5 ± 1.7 Ma, which overlaps the emplacement of proximal post-tectonic granitoid magmatism. Additionally, we suggest that arsenopyrite growth may have occurred over an interval of at least 2 m.y. Os initial ratios derived from arsenopyrite were coupled with He isotopic data from fluid inclusions within arsenopyrite to constrain the source of ore metals and fluids. Muruntau arsenopyrite yields relatively unradiogenic initial Os (0.37 ± 0.27) and elevated 3He/4He ratios (0.23–0.33Ra) relative to purely crustal Os-He reservoirs. These data suggest the presence of a mantle-derived component in the ore system that was probably introduced during the generation of the granitoid magmas. These new timing and source constraints provide important new insight into the generation of this giant gold deposit, and they necessitate reexamination of genetic models for Muruntau and potentially other giant “orogenic gold” deposits worldwide.