In response to brain injury, microglia migrate and accumulate in the affected sites, which is an important step in the regulation of inflammation and neuronal degeneration/regeneration. In this study, we investigated the effect of urokinase-type plasminogen activator (uPA) on the BV-2 microglial cell migration. At resting state, BV-2 microglial cells secreted uPA and the release of uPA was increased by ATP, a chemoattractant released from injured neuron. The migration of BV-2 cell was significantly induced by uPA and inhibited by uPA inhibitors. In this condition, uPA increased the activity of matrix metalloproteinase (MMP-9) and the inhibition of MMP activity with pharmacological inhibitors against either uPA (amiloride) or MMP (phenanthrolene and SB-3CT) effectively prevented BV2 cell migration. Interestingly, the level of MMP-9 protein and mRNA in the cell were not changed by uPA. These results suggest that the increase of MMP-9 activity by uPA is regulated at the post-translational level, possibly via increased activation of the enzyme. Unlike the uPA inhibitor, plasmin inhibitor PAI-1 only partially inhibited uPA-induced cell migration and MMP-9 activation. The incubation of recombinant MMP-9 with uPA resulted in the activation of MMP-9. These results suggest that uPA plays a critical role in BV-2 microglial cell migration by activating pro-MMP-9, in part by its direct action on MMP-9 and also in part by the activation of plasminogen/plasmin cascade.