Microbially mediated iron (Fe) reduction is suggested to be one of the earliest metabolic pathways on Earth and Fe(III)-reducing microorganisms might be key inhabitants of the deep and hot biosphere [1, 2]. Since microbial Fe cycling is typically accompanied by Fe isotope fractionation, stable Fe isotopes (δ56Fe) are used as tracer for microbial processes in modern and ancient marine sediments [3, 4]. Here we present Fe isotope data for dissolved and sequentially extracted sedimentary Fe pools from subseafloor sediments that were recovered during International Ocean Discovery Program Expedition 370 from a 1,180 m deep hole drilled in the Nankai Trough off Japan where temperatures of up to 120°C are reached at the sediment-basement interface. The expedition aimed at exploring the temperature limit of microbial life and identifying geochemical and microbial signatures that differentiate the biotic and abiotic realms [5, 6]. Dissolved Fe (Fe(II)aq) is isotopically light throughout the ferruginous sediment interval but some samples have exceptionally light δ56Fe values. Such light δ56Fe values have never been reported in natural marine environments and cannot be solely attributed to microbially mediated Fe(III) reduction. We show that the light δ56Fe values are best explained by a Rayleigh distillation model where Fe(II)aq is continuously removed from the pore water by diffusion and adsorption onto Fe (oxyhydr)oxide surfaces. While the microbially mediated Fe(II)aq release has ceased due to an increase in temperature beyond the threshold of mesophilic microorganisms, the abiotic diffusional and adsorptive Fe(II)aq removal continued, leading to uniquely light δ56Fe values. These findings have important implications for the interpretation of Fe isotope records especially in deep subseafloor sediments.