The Early Paleozoic arc-hosted volcanogenic massive sulfide (VMS) deposits in the Kalatag arc are well-known for their well-preserved and high-grade Cu–Zn sulfide mineralization in the East Tianshan. Unfortunately, there is a puzzle on the ore-forming fluid and metal source as well as physicochemical condition of metallogenic process of these arc-hosted VMS Cu–Zn deposits. The Huangtupo was firstly reported Early Paleozoic arc-hosted VMS Cu–Zn deposit (300Kt@1.49 % Cu; 280Kt@3.51 % Zn) in the East Tianshan. In order to better recognize the physicochemical condition and formation processe of arc-hosted VMS deposits, this research takes the Huangtupo deposit as an example, combining the microstructure, in-situ sulfur isotope and trace element composition of multistage sulfides to conduct a comprehensive study. In the Huangtupo deposit, the overall mineralization process is divided into syndepositional mineralization stage, hanging-wall massive mineralization stage and channel hydrothermal stage according to the main mineral interpenetration relationship and horizon relationship. Pyrite can be divided into five types in the Huangtupo Cu–Zn deposit: 1) subhedral to anhedral pyrite in the silicious exhalite (Py1); 2) colloform pyrite in the hanging-wall massive pyrite + barite + sphalerite zone (Py2); 3) subhedral pyrite in the hanging-wall massive pyrite + chalcopyrite + sphalerite ± magnetite zone (Py3); 4) fine-grained (or disseminated) pyrite in the footwall pyrite + chalcopyrite + sphalerite stage (Py4); 5) euhedral pyrite in the footwall quartz + pyrite + chalcopyrite stage (Py5). Two types of chalcopyrite are identified, including hanging-wall massive chalcopyrite (Ccp1) and footwall stockwork chalcopyrite (Ccp2). In addition, three generations of sphalerite could be identified, namely (1) hanging-wall massive pyrite + barite + sphalerite zone (Sp1); (2) hanging-wall massive pyrite + chalcopyrite + sphalerite ± magnetite zone (Sp2); and (3) footwall pyrite + quartz + sphalerite + chalcopyrite stage (Sp3). In the upper massive orebody, LA–ICP–MS in-situ microanalysis shows that pyrite is enriched in Cu, Zn, Au, Ag, Pb, Sb, and Tl, while it is poor in Co, Ni, Se, Te, Ti, and Sn, indicating that it was formed in a metal-rich, medium–high temperature and more oxidizing hydrothermal environment. By contrast, pyrite from the footwall stockwork orebody contains low Au, Ag, Cu, Zn, Pb, Sb, Tl and high Co, Se, Te, Ti content, indicating that it is formed in the hydrothermal environment with a high temperature, reduction and trace metal deficit signature. Trace element compositions of sphalerite suggest that Fe, Mn, Ag, Se, In, Ge, and Ga are present as lattice. By contrast, Sn, Pb, Tl, and Bi are present as mineral microinclusions. The mineralization temperature is determined by sphalerite geothermometer (GGIMFis) and shows a decreasing trend from footwall stockwork mineralization to hanging-wall massive mineralization. Comparing with sphalerite, chalcopyrite is enriched in Ge, Ag, Zn, Se, Sn, and Cd, relatively poor in In and Ga. The integrated in situ sulfur isotopic (δ34S) composition and trace metal content of sulfides suggest that ore-forming material is most likely derived from a combination of host volcanics and seawater. In addition, the thermochemical sulfate reduction is also a key factor affecting sulfur isotope fractionation. Overall, the trace element compositions of multistage sulfides are robust monitor of the complex hydrothermal process and effectively reconstruct the metal enrichment process during the formation of arc-hosted VMS mineralization system.