Disruption of cell contact sites in renal epithelial cells contributes to organ dysfunction after ischemia. We hypothesized that heat shock protein 27 (Hsp27), a known cytoprotectant protein, preserves cell architecture and cell contact site function during ischemic stress. To test this hypothesis, renal epithelial cells were subjected to transient ATP depletion, an in vitro model of ischemia-reperfusion injury. Compared with control, selective Hsp27 overexpression significantly preserved cell-cell junction function during metabolic stress as evidenced by reduced stress-mediated redistribution of the adherens junction protein E-cadherin, higher transepithelial electrical resistance, and lower unidirectional flux of lucifer yellow. Hsp27 overexpression also preserved paxillin staining within focal adhesion complexes and significantly decreased cell detachment during stress. Surprisingly, Hsp27, an F-actin-capping protein, only minimally reduced stress induced actin cytoskeleton collapse. In contrast to Hsp27 overexpression, siRNA-mediated knockdown had the opposite effect on these parameters. Since ischemia activates c-Src, a tyrosine kinase that disrupts both cell-cell and cell-substrate interactions, the relationship between Hsp27 and c-Src was examined. Although Hsp27 and c-Src did not coimmunoprecipitate and Hsp27 overexpression failed to inhibit whole cell c-Src activation during injury, manipulation of Hsp27 altered active c-Src accumulation at cell contact sites. Specifically, Hsp27 overexpression reduced, whereas Hsp27 knockdown increased active p-(416)Src detected at contact sites in intact cells as well as in a purified cell membrane fraction. Together, this evidence shows that Hsp27 overexpression prevents sublethal REC injury at cell contact sites possibly by a c-Src-dependent mechanism. Further exploration of the biochemical link between Hsp27 and c-Src could yield therapeutic interventions for ameliorating ischemic renal cell injury and organ dysfunction.