Renal ischemia and in vitro ATP-depletion result in disruption of the epithelial tight junction barrier, which is accompanied by breakdown of plasma membrane polarity. Tight junction formation is regulated by evolutionarily conserved complexes, including that of atypical protein kinase C (aPKC), Par3 and Par6. The aPKC signaling complex is activated by Rac and regulated by protein phosphorylation and associations with other tight junction regulatory proteins, for example mLgl. In this study, we examined the role of aPKC signaling complex during ATP-depletion and recovery in Madin-Darby canine kidney (MDCK) cells. ATP-depletion reduced Rac GTPase activity and induced Par3, aPKC and mLgl-1 redistribution from sites of cell-cell contact, which was restored following recovery from ATP-depletion. ZO-1 and Par3 phosphorylation was reduced and association of aPKC with its substrates Par3 and mLgl-1 was stabilized in ATP-depleted MDCK cells. ATP-depletion also induced a stable association of Par3 with Tiam-1, a Rac GTPase exchange factor (GEF), which explains how aPKC and Rac activities were suppressed. Experimental inhibition of aPKC during recovery from ATP-depletion interfered with reassembly of ZO-1 and Par3 at cell junctions. These data indicate that aPKC signaling is impaired during ATP-depletion, participates in tight junction disassembly during cell injury and is important for tight junction reassembly during recovery.