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Equilibrium isotope fractionation factors are the basis for application of stable isotopes in geological studies. Experimental calibration and theoretical calculations have been employed to determine Fe isotope fractionation factors for a variety of minerals, however, these methods have their limitations. An alternative approach to calibrating inter-mineral Fe isotope fractionation factors is to use well-characterized geological samples; this approach has unique advantages over the other methods including attainment of equilibrium at relatively low temperatures. In this study, we investigated the Fe isotope composition of magnetite (Mt), garnet (Grt), amphibole (Amp) and biotite (Bt) from the metamorphosed Yingshan iron formation in South China. Two independent geothermometers, quartz–magnetite oxygen isotope and amphibole–garnet–biotite Fe–Mg exchange geothermometers, give a consistent metamorphic temperature of 538 ± 39 °C. The Fe isotope composition of the different Fe-bearing minerals is highly variable in different ironstone samples, with δ56Fe values (relative to IRMM-014) ranging from −0.23 to +0.37‰ in magnetite, −0.44 to +0.09‰ in amphibole, −0.78 to +0.02‰ in garnet, and −0.61 to +0.04‰ in biotite. Despite that, the offsets of δ56Fe values for mineral pairs are consistent, implying attainment of equilibrium isotope fraction between these minerals. Inter-mineral Fe isotopic fractionations (±2 standard deviation) measured from the multiple mineral pairs are Δ56FeMt–Grt = +0.55 ± 0.08‰, Δ56FeMt–Amp = +0.25 ± 0.06‰, Δ56FeMt–Bt = +0.42 ± 0.11‰, Δ56FeAmp–Grt = +0.34 ± 0.15‰, Δ56FeAmp–Bt = +0.26 ± 0.04‰, and Δ56FeBt–Grt = +0.13 ± 0.08‰. Based on the well-defined metamorphic temperature (538 ± 39 °C) and internally consistent inter-mineral fractionation factors, the temperature-dependent functions for equilibrium Fe isotope fractionation between the following mineral pairs are derived: 103lnαMt–Grt = 0.36(±0.05) × 106/T2, 103lnαMt–Bt = 0.28(±0.07) × 106/T2, 103lnαMt–Amp = 0.16(±0.04) × 106/T2, 103lnαAmp–Grt = 0.22(±0.10) × 106/T2, 103lnαAmp–Bt = 0.17(±0.03) × 106/T2, and 103lnαBt–Grt = 0.09(±0.05) × 106/T2, where Fe3+/ΣFe ratio is 0.05 ± 0.02 in garnet, 0.29 ± 0.04 in biotite, and 0.24 ± 0.06 in amphibole for the above functions. The equilibrium Fe isotope fractionation factors derived in this study enable estimation of the metamorphic temperature of rocks that contain these mineral pairs, and identification of secondary processes that may have induced disequilibrium Fe isotope distribution in rocks, such as retrograde metamorphism and hydrothermal alteration.