This study presents experimental copper isotope (65Cu/63Cu) fractionation data between chalcopyrite and dissolved Cu at physiochemical conditions representative of high temperature mid-ocean ridge hydrothermal environments. The experimental data are compared with chemical and Cu isotope data from high temperature fluids and chalcopyrite sampled from the Main Endeavour Field and ASHES hydrothermal fields located along the Juan de Fuca Ridge. Specifically, three long-term ( > 250-2500 hr) chalcopyrite recrystallization experiments were conducted at 350 °C and 50 MPa in acidic chloride-bearing fluid. One of the experiments contained 57Fe isotope tracer to quantify reaction progress and the extent of exchange between chalcopyrite and dissolved Fe, which serves as a proxy for Cu isotope exchange (Syverson et al., 2017). The dissolved 57Fe tracer demonstrated rapid isotope exchange between chalcopyrite and dissolved metals within the duration of the long term experiments, approximately 2500 hr, where complete exchange was achieved within approximately 1000 hr. The experimentally determined δ65Cu equilibrium fractionation between chalcopyrite and dissolved Cu,Δ65Cu[Cpy-Cuaq] = −0.22 ± 0.16‰ (1σ), is comparable in sign and magnitude to theoretical predictions. Comparison of the δ65Cu values of chimney-derived chalcopyrite and hydrothermal fluids sampled from the Main Endeavour Field and ASHES hydrothermal systems demonstrate a range in (dis)equilibrium from theoretical and experimental Δ65Cu[Cpy-Cuaq] constraints, possibly due to temporal fluctuations in the δ65Cu value of hydrothermal fluid associated with changes in the physiochemical processes controlling Cu mass transfer in the subseafloor. Overall, the small Δ65Cu[Cpy-Cuaq] effectively allows chalcopyrite to be a reliable recorder of the variability in the δ65Cu value of end-member hydrothermal fluids throughout the temporal evolution of MOR hydrothermal systems.