The Huangtupo Cu-Zn (-Au) volcanogenic massive sulfide deposit is hosted in a suite of Early Paleozoic basic to acidic volcanic, volcaniclastic rocks in the Eastern Tianshan orogen, northwest China. The Cu-Zn orebodies are characterized by the zoning of metallogenic elements upward from a deep stockwork mineralized zone (Cu > Zn) to a shallow massive mineralized zone (Zn > Cu). Detailed petrographic and LA-ICP-MS analyses have identified five types of pyrite and three types of sphalerite. These sulfides can be distinguished in a paragenesis with four stages of sulfide mineralization. The preore stage (SⅠ) from the deep volcanic rock contains subhedral-euhedral pyrite (Py1) with high trace element contents (e.g., Co, Ni, and Ti; Average value of total trace elements: 16764 ppm), except for Sb, and Tl, indicating a euxinic and high-temperature environment. Both the disseminated and veined pyrite (Py2) from the early ore stage (SⅡ; stockwork zone) and the euhedral pyrite (Py3) from the main ore stage-1 (SIII-1) have a similar chemical signature, suggesting that they precipitated under similar physicochemical conditions. These two paragentic stages of pyrite contributed only minimally to the trace elements endowment, except for higher Se contents. The continuous convection of hydrothermal fluids caused a large accumulation of copper and zinc, forming massive orebodies. Pyrite within the Zn(Cu)-rich zone of the main ore stage-2 (SIII-2) comprises three sub-generations (Py4a ∼ c) which are characterized by enrichment in most trace elements such as Au, As, Cu, and Sb. Pyrite 4a ∼ c formed in a fluctuating sedimentary environment and recorded the injection of a short-term, gold-rich fluid, which dissolved and reprecipitated trace elements from earlier ores and surrounding rocks. Orebody deformation and local metamorphism played a key role in this gold enrichment event. Pyrite (Py5) from the late ore stage (SⅣ) is deficient in most elements but relatively enriched in Co, Ni, and Se. Three stages of sphalerite associated with the Py3 ∼ 4 have been distinguished in the SIII. From Sp1 to Sp3, the content of Fe, Ge, Mo, and Cd decreased gradually, but the Sp2 is relatively depleted in Ag, As, Au, Pb, and Sb compared to Sp1 and Sp3. The sensitive variation of As, Co, Ni, Se, Pb in pyrite and Fe, Cu, Mn, Sb, Cd in sphalerite provide significant information for reconstructing the hydrothermal evolution process. The sulfur isotopes of these pyrites determined by in situ LA-multicollector (MC)-ICP-MS analyses are concentrated around 0‰, but vary significantly from −2.3 to 5.8‰. The δ34S values of the sphalerite range from −3.5 to 4.0 ‰ and tends to decrease from Sp1 to Sp3. These results show that the metals of the SIII were most likely derived from the deep-seated Early Paleozoic subvolcanic intrusion and Daliugou Formation during the evolution of regional island arcs.Combining the chemical, and isotopic data and geological features, the ore-forming process of an ancient VMS deposit was deduced. These results constrain the texture, composition and sulfur isotopic signatures of sulfides in veins and deformed ore bodies, as well as the gold enrichment mechanism, and also reflect the vertical variation of trace elements at the scale of VMS deposits, supporting the existence of multiple depositional mechanisms.