Disruption of cell contact sites in renal epithelial cells contributes to organ dysfunction after ischemia. We hypothesized that 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 to control, selective Hsp27 over-expression significantly preserved cell-cell junction function during metabolic stress as evidenced by reduced stress mediated re-distribution of the adherens junction protein E-cadherin, higher transepithelial electrical resistance and lower unidirectional flux of lucifer yellow. Hsp27 over-expression 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 over-expression, 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 co-immunoprecipitate and Hsp27 over-expression failed to inhibit whole cell c-Src activation during injury, manipulation of Hsp27 altered active c-Src accumulation at cell contact sites. Specifically, Hsp27 over-expression reduced, whereas Hsp27 knockdown increased active p-⁴¹⁶Src detected at contact sites in intact cells as well as in a purified cell membrane fraction. Together, this evidence shows that Hsp27 over-expression 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.