In this study, we report on coupled Fe- and S-isotope systematics of hydrothermal fluids and sulfide deposits from the East Pacific Rise at 9–10°N to better constrain processes affecting Fe-isotope fractionation in hydrothermal environments. We aim to address three fundamental questions: (1) Is there significant Fe-isotope fractionation during sulfide precipitation? (2) Is there significant variability of Fe-isotope composition of the hydrothermal fluids reflecting sulfide precipitation in subsurface environments? (3) Are there any systematics between Fe- and S-isotopes in sulfide minerals? The results show that chalcopyrite, precipitating in the interior wall of a hydrothermal chimney displays a limited range of δ⁵⁶Fe values and δ³⁴S values, between − 0.11 to − 0.33‰ and 2.2 to 2.6‰ respectively. The δ⁵⁶Fe values are, on average, slightly higher by 0.14‰ relative to coeval vent fluid composition while δ³⁴S values suggest significant S-isotope fractionation (− 0.6 ± 0.2‰) during chalcopyrite precipitation. In contrast, systematically lower δ⁵⁶Fe and δ³⁴S values relative to hydrothermal fluids, by up to 0.91‰ and 2.0‰ respectively, are observed in pyrite and marcasite precipitating in the interior of active chimneys. These results suggest isotope disequilibrium in both Fe- and S-isotopes due to S-isotopic exchange between hydrothermal H₂S and seawater SO₄²⁻ followed by rapid formation of pyrite from FeS precursors, thus preserving the effects of a strong kinetic Fe-isotope fractionation during FeS precipitation. In contrast, δ⁵⁶Fe and δ³⁴S values of pyrite from inactive massive sulfides, which show evidence of extensive late-stage reworking, are essentially similar to the hydrothermal fluids. Multiple stages of remineralization of ancient chimney deposits at the seafloor appear to produce minimal Fe-isotope fractionation. Similar affects are indicated during subsurface sulfide precipitation as demonstrated by the lack of systematic differences between δ⁵⁶Fe values in both high-temperature, Fe-rich black smokers and lower-temperature, Fe-depleted vents.