Meaningful interpretation of U isotope measurements relies on unraveling the impact of reduction mechanisms on the isotopic fractionation. Here, the isotope fractionation of hexavalent U [U(VI)] was investigated during its reductive mineralization by magnetite to intermediate pentavalent U [U(V)] and ultimately tetravalent U [U(IV)]. As the reaction proceeded, the remaining aqueous phase U [containing U(VI) and U(V)] systematically carried light isotopes, whereas in the bicarbonate-extracted solution [containing U(VI) and U(V)], the delta U-238 values varied, especially when C/C-0 approached 0. This variation was interpreted as reflecting the variable relative contribution of unreduced U(VI) (delta U-238 < 0 parts per thousand) and bicarbonate-extractable U(V) (delta U-238 > 0 parts per thousand). The solid remaining after bicarbonate extraction included unextractable U(V) and U(IV), for which the delta U-238 values consistently followed the same trend that started at 0.3-0.5 parts per thousand and decreased to similar to 0 parts per thousand. The impact of PIPES buffer on isotopic fractionation was attributed to the variable abundance of U(V) in the aqueous phase. A few extremely heavy bicarbonate-extracted delta U-238 values were due to mass-dependent fractionation resulting from several hypothesized mechanisms. The results suggest the preferential accumulation of the heavy isotope in the reduced species and the significant influence of U(V) on the overall isotopic fractionation, providing insight into the U isotope fractionation behavior during its abiotic reduction process.