The early Paleozoic North Qilian Orogenic Belt (NQOB) records a complex history, from early oceanic subduction and back-arc extension to the eventual closure of the ocean and continental collision. The tectonic nature of the back-arc basin and its evolution are debated. The Mijiashan (MJS) ophiolite in the eastern NQOB is thought to represent remnants of the North Qilian back-arc, and consists mainly of a crustal sequence of basalts, plagiogranites, and gabbroic intrusions. A fine-grained gabbroic diorite intrusion has recently been identified in this crustal sequence. We present new zircon U–Pb ages and bulk-rock chemical and Sr–Nd isotopic data for these rocks. Zircon grains from the gabbroic diorite yield a U–Pb age of 510 Ma, which is taken to be the crystallization age of the rocks, consistent with the field relationship. The diorite is tholeiitic in composition, enriched in Cs, Ba, Sr, and Pb, and depleted in Nb, Ta, Th, and Rb on a primitive-mantle-normalized trace element spider diagram. It has low total rare earth element (REE) contents and yields chondrite-normalized rare earth element patterns depleted in light REEs. The MJS gabbroic diorite has depleted isotopic compositions [whole-rock ε_Nd(t) = +4.96 to + 5.25; initial ⁸⁷Sr/⁸⁶Sr ratio = 0.7055–0.7066]. These geochemical and isotopic characteristics suggest that these rocks were formed from melts that were derived from partial melting of residual, extremely refractory, hydrated mantle peridotite. This mantle wedge source had previously experienced the extraction of MORB-type melts and was subsequently metasomatized primarily by subduction-derived fluids, with minimal contributions from seafloor sediment components. The gabbroic diorite of the MJS ophiolite was formed in an intracontinental back-arc setting and represents the earliest identified magmatic record of the initiation of arc rifting in a supra-subduction zone environment in response to rollback of the northward-subducting North Qilian oceanic lithosphere.