The two-step electron transfer during bacterial reduction of UVI to UIV is typically accompanied by mass-independent fractionation of the 238U and 235U isotopes, whereby the heavy isotope accumulates in the reduced product. However, the role of the UV intermediate in the fractionation mechanism is unresolved due to the challenges associated with its chemical stability. Here, we employed the UV stabilizing ligand, dpaea2−, to trap aqueous UV during UVI reduction by Shewanella oneidensis. Whilst the first reduction step from UVI to UV displayed negligible fractionation, reduction of UV to UIV revealed mass-dependent isotope fractionation (preferential reduction of the 235U), contrary to most previous observations. This surprising behaviour highlights the control the U-coordinating ligand exerts over the balance between reactant U supply, electron transfer rate, and UIV product sequestration, suggesting that UV speciation should be considered when using U isotope ratios to reconstruct environmental redox conditions.