The D” layer at the base of the Earth’s mantle exhibits anomalous seismic properties, which are attributed to heat loss from and chemical interaction with the underlying molten Fe-rich outer core. Here we show that mass transfer due to temperature variations within the D” layer could lead to resolvable fractionation of iron isotopes. We constrain the degree of isotope fractionation by experiments on core-forming Fe alloy liquids at 2100–2300 K and 2 GPa, which demonstrate that heavy Fe isotopes preferentially migrate towards lower temperature and vice versa. We find that this isotope fractionation occurs rapidly due to the high mobility of iron, which reaches 0.013 ± 0.002‰ (2σ) per degree per amu at steady state. Numerical simulations of mantle convection capturing the evolution of a basal thermal boundary layer show that iron isotope fractionation immediately above the core–mantle boundary can reach measurable levels on geologic timescales and that plumes can entrain this fractionated material into the convecting mantle. We suggest that such a process may contribute to the heavy Fe isotope composition of the upper mantle inferred from mantle melts (basalts) and residues (peridotites) relative to chondrites. That being the case, non-traditional stable isotope systems such as Fe may constrain the interactions between the core and mantle.