Studies on the Fe, Cu, and Zn isotopic compositions of volcanic rocks and sulfides provide an important tool for understanding magmatic, hydrothermal, and alteration processes. In this study, the δ56Fe and δ57Fe values of the MORBs are higher than those of the seafloor hydrothermal fluids, while the reverse is true for the δ66Zn and δ68Zn values, suggesting that basalt-fluid interactions preferentially incorporate isotopically light Fe and heavy Zn into the fluid, resulting in the relative enrichment of the heavier Fe and lighter Zn isotopes in altered basaltic rocks. Most of the δ56Fe values (–1.96 to +0.11‰) of the sulfide minerals are within the range of the vent fluids, but they are significantly lower than those of MORBs and back-arc basin basalts (BABBs), suggesting that the Fe in the sulfides was mainly derived from the fluids. However, the majority of the δ56Fe and δ57Fe values of chalcopyrite are larger than those of sphalerite and pyrite. This suggests that high-temperature sulfide minerals are enriched in 56Fe and 57Fe, whereas medium- and low-temperature sulfides are depleted in 56Fe and 57Fe. Moreover, the δ65Cu (–0.88 to –0.16‰) and δ66Zn (–0.39 to –0.03‰) values of the sulfide minerals are significantly lower than those of the MORBs, BABBs, and fluids, suggesting that 63Cu and 64Zn were preferentially removed from the fluids and incorporated into the chalcopyrite and sphalerite, respectively. Consequently, vent fluid injection and deposition can cause the heavier Cu and Zn isotopic compositions of hydrothermal plumes, seawater, and sediments.