The infectivity of HIV-1 virions can be enhanced by inhibition of the proteasome in target cells, leading to the hypothesis that the proteasome degrades incoming virions as part of the intracellular antiviral defense. Here, several lines of evidence suggest instead that proteasome inhibition renders target cells more susceptible to infection via an indirect effect on the cellular environment: (1) proteasome inhibition increased infectivity more effectively when target cells were exposed to the inhibitors before exposure to virions, rather than when the inhibitors and virions were present simultaneously; (2) increased infectivity correlated directly with the duration of pre-exposure of cells to the inhibitors; (3) although increased infectivity was induced by as little as 30 min of pretreatment of target cells, binding of virions to target cells before the addition of inhibitor abolished the effect; and (4) increased infectivity persisted after removal of the inhibitors and the recovery of proteasome activity within the target cells. Cell cycle analyses revealed that an increased fraction of cells in G2/M may correlate with increased efficiency of infection. These data suggest that rather than relieving a target cell restriction based on the degradation of incoming virions, proteasome inhibitors likely increase infectivity either via their effects on the cell cycle or by increasing the expression of a host cell factor that facilitates infection.