The crustal and upper mantle structure of the Beishan orogenic collage, which serves as the southern part of the middle Central Asian Orogenic Belt, provides crucial insights into the history of the multiple openings and closings of the Paleo-Asian Ocean during the Paleozoic. There is considerable dispute over the eventual closure position, timing, and subduction polarity of the Paleo-Asian Ocean, particularly in the southern Beishan orogenic collage. The main cause of these controversies is the lack of a high-resolution lithospheric structure in this area. In this study, we first present a 140-km-long, high-resolution seismic reflection profile taken across the northern Dunhuang Block and the southern Beishan orogenic collage. The seismic imaging provides new constraints on the structure of the lower crust, the Moho, and the upper mantle beneath the southernmost Central Asian Orogenic Belt. A subhorizontal reflector in the middle crust, two sets of north-dipping reflectors from the lower crust to the upper mantle, and several south-dipping reflectors in the upper crust in the northern part of the profile were imaged. Based on our study and other geological, chronological, and geophysical data, we propose that the two sequences of north-dipping reflectors from the lower crust to the upper mantle represent two stages of north-dipping subduction of the southern branch of the Paleo-Asian Ocean. The first stage in the southern Beishan orogenic collage is Late Silurian−Early Devonian, and the second stage is late Carboniferous−Early Permian. The two-stage subduction process gave rise to the Huaniushan arc and the subsequent Shibanshan arc, respectively. These findings provide new constraints on the controversial subduction polarity and the multistage amalgamation of the microcontinental blocks and arcs in the southern Central Asian Orogenic Belt.