The RNA-binding protein HuR participates in the post-transcriptional regulation of mRNAs bearing 3' AU-rich and U-rich elements, which HuR can stabilize under conditions of cellular stress. Using the LLC-PK₁ proximal tubule cell line model we recently suggested a role for HuR in protecting kidney epithelia from injury during ischemic stress. Here we have extended this work to show that siRNA-mediated suppression of HuR in LLC-PK₁ cells increased apoptosis during energy depletion, while overexpression of HuR diminished apoptosis. Suppression of HuR also resulted in diminished levels of key cell survival proteins such as Bcl-2 and Hsp70. Further, rat kidneys were subjected in vivo to transient ischemia followed by varying periods of reperfusion. Ischemia and reperfusion (I/R) affected intensity and distribution of HuR in a nephron segment-specific manner. Cells of the proximal tubule, which are most sensitive to I/R injury, demonstrated a transient shift of HuR to the cytoplasm immediately following ischemia. Over a 14-day period following the onset of reperfusion, nuclear and total HuR protein gradually increased in cortical and medullary proximal tubules, but not in non-proximal tubule cells. HuR mRNA was expressed in two forms with alternate transcriptional start sites that increased over a 14-day ischemia-reperfusion period, and in vitro studies suggest selective translatability of these two mRNAs. Baseline and I/R-stimulated levels of HuR mRNA did not parallel those of HuR protein, suggesting translational control of HuR expression, particularly in medullary proximal tubules. These findings suggest that alterations in distribution and expression of the anti-apoptotic protein HuR specifically in cells of the proximal tubule effects a protective mechanism during and following I/R injury in kidney.