The Fe isotope fractionation factors among aqueous ferrous iron (Feaq2+), aqueous FeS clusters (FeSaq), and nanoparticulate mackinawite under neutral and mildly acidic and alkaline pH conditions have been determined using the three-isotope method. Combined voltammetric analysis and geochemical modeling were used to determine the Fe speciation in the experimental systems. The equilibrium 56Fe/54Fe fractionation factor at 20 °C and pH 7 has been determined to be −0.32 ± 0.29 (2σ)‰ between Feaq2+ (minor FeSaq also present in the experiment) and mackinawite. This fractionation factor was essentially constant when pH was changed to 6 or 8. When equal molarity of HS− and Feaq2+ were added to the system, however, the isotopic fractionation at pH 7 changed to −0.64 ± 0.36 (2σ)‰, correlating with a significant increase in the proportion of FeHS+ and FeSaq. These results highlight a more important role of aqueous Fe–S speciation in the equilibrium Fe isotope fractionation factor than recognized in previous studies. The isotopic fractionation remained constant when temperature was increased from 20 °C to 35 °C for fractionation factors between Feaq2+, and mackinawite and between dominantly FeHS+ and mackinawite. Synthesis experiments similar to those of Butler et al. (2005) and Guilbaud et al. (2010) at pH 4 show consistent results: over time, the aqueous Fe–mackinawite fractionation decreases but even after 38 days of aging the fractionation factor is far from the equilibrium value inferred using the three-isotope method. In contrast, at near-neutral pH the fractionation factor for the synthesis experiment reached the equilibrium value in 38 days. These differences are best explained by noting that at low pH the FeS mackinawite particles coarsen more rapidly via particle aggregation, which limits isotopic exchange, whereas at higher pH mackinawite aggregation is limited, and Fe isotope exchange occurs more rapidly, converging on the equilibrium value. These results suggest that mackinawite formed in natural environments at near-neutral or alkaline pH are unlikely to retain kinetic isotope fractionations, but are more likely to reflect equilibrium isotope compositions. This in turn has important implications for interpreting iron isotope compositions of Fe sulfides in natural systems.