The Hongtoushan Volcanogenic Massive Sulphide Deposit (VMSD) occurs in the Hunbei granite-greenstone terrane, Liaoning Province, NE China. Rocks in the mining area have been metamorphosed around 3.0–2.8 Ga to upper amphibolite facies at temperatures between 600°C and 650°C. Cordierite-anthophyllite gneiss (CAG) in the Hongtoushan mining area, which occurs hundreds of meters below the ore horizon, corresponds to the metamorphosed semi-conformable alteration zone of the VMSD hydrothermal system, whereas the one immediately below the main ore layer represents the metamorphosed pipe-like alteration zone. Whole-rock oxygen isotope signatures were well preserved in both types of CAGs, although the mineral components have been entirely changed during regional metamorphism. Therefore, whole-rock oxygen isotopes can be used to estimate the formation temperature of both types of alteration zone. Calculations show that the semi-conformable and pipe-like alteration zones for the Hongtoushan submarine hydrothermal system were formed at 290–360°C and 285–320°C, respectively, whereas estimates for the former were slightly higher than that of the latter, indicating that the semi-conformable alteration zone represents the deep part of the Hongtoushan seafloor hydrothermal system, while the pipe-like alteration zone represents the discharge conduits for metal-rich fluids, which is closer to the seafloor.