Human UDP-glucose dehydrogenase (hUGDH) catalyzes the oxidation of UDP-glucose into UDP-glucuronic acid, an essential substrate in the Phase II metabolism of drugs. hUGDH is a hexamer that exists in an equilibrium between an active (E) state and an inactive (EΩ) state, with the latter being stabilized by the binding of the allosteric inhibitor UDP-xylose (UDP-Xyl). The allosteric transition between EΩ and E is slow and can be observed as a lag in progress curves. Previous analysis of the lag suggested that unliganded hUGDH exists mainly as EΩ, but two unique crystal forms suggest that the enzyme favors the E state. Resolving this discrepancy is necessary to fully understand the allosteric mechanism of hUGDH. Here, we used cryo-EM to show that recombinant hUGDH expressed in Escherichia coli copurifies with UDP-4-keto-xylose (UX4O), which mimics the UDP-Xyl inhibitor and favors the EΩ state. Cryo-EM studies show that removing UX4O from hUGDH shifts the ensemble to favor the E state. This shift is consistent with progress curve analysis, which shows the absence of a lag for unliganded hUGDH. Inhibition studies show that hUGDH has similar affinities for UDP-Xyl and UX4O. The discovery that UX4O inhibits allosteric hUGDH suggests that UX4O may be the physiologically relevant inhibitor of allosteric UGDHs in bacteria that do not make UDP-Xyl.