Uranium contamination in the environment is a serious public health concern. Biotic U(VI) reduction and nonreductive U(VI) uptake by microorganisms (e.g., U(VI) biosorption by cyanobacteria) are effective U remediation techniques. Variations of 238U/235U have been extensively explored to track biotic U(VI) reduction in laboratory experiments and field applications. However, U isotope fractionation during nonreductive U(VI) uptake by microorganisms is poorly constrained. To investigate U isotope fractionation in this process, we cultured freshwater plankton in the presence of U(VI) and measured 238U/235U in the culture media and biomass. We found that nonreductive U(VI) uptake by freshwater plankton fractionated U isotopes in the opposite direction compared to biotic U(VI) reduction. δ238U values in freshwater plankton were consistently ∼0.23 ± 0.06‰ lighter than those in dissolved U in the culture medium at various fractions of U removal (12-30%), consistent with equilibrium isotope fractionation in a closed system. The equilibrium isotope fractionation observed in our experiments possibly results from changes in coordination geometry between dissolved U(VI) in the culture media and adsorbed U(VI) on cell surfaces. Our experimental results highlight the need to consider U isotope fractionation during nonredox U(VI) uptake by microorganisms and organic matter when applying variations of 238U/235U to track biogeochemical processes and evaluate U remediation.