Abstract Reduction of Cr(VI) is an important process that determines the geochemical behavior, mobility and bioavailability of Cr in both terrestrial and marine environments. Many metabolically diverse microorganisms possess Cr(VI) reduction capacity. Cr(VI) reduction fractionates Cr isotopes and thus 53Cr/52Cr ratios can be used to monitor Cr(VI) reduction and redox conditions. The magnitude of isotopic fractionation (e) for a variety of microbial reduction mechanisms must be known for accurate interpretation of observed shifts in 53Cr/52Cr ratios. We determined isotopic fractionation factors for Cr(VI) reduction by metal reducers Geobacter sulfurreducens and Shewanella sp. strain NR, a denitrifying soil bacterium Pseudomonas stutzeri DCP-Ps1, and a sulfate reducer Desulfovibrio vulgaris. All bacteria investigated in this study produced significant Cr isotope fractionation. The fractionation (e) for G. sulfurreducens, Shewanella sp. (NR), P. stutzeri DCP-Ps1, and D. vulgaris were −3.03‰ ± 0.12‰, −2.17‰ ± 0.22‰, −3.14‰ ± 0.13‰, and −3.01‰ ± 0.11‰, respectively. Despite differences in microbial strains in this study, the e did not vary significantly except for Shewanella sp. (NR). Our results suggest that strong isotopic fractionation is induced during Cr(VI) reduction under electron donor poor (∼300 μM) conditions.