In previous studies we demonstrated that the presence of testosterone, rather than the absence of estrogen, plays a critical role in gender differences in kidney ischemia/reperfusion (I/R) injury. Although molecular chaperones such as heat shock proteins (HSPs) have been implicated as protective agents in the pathophysiology of I/R injury, their roles in gender differences in susceptibility to renal I/R injury remain to be defined. Here we demonstrate that orchiectomy increases the basal and post-ischemic expression of HSP-27 in kidney tubular epithelial cells, but not HSP-72, glucose-regulated protein (GRP)-78 or GRP-94 expression. Orchiectomy prevents the disruption of the actin cytoskeleton and renal functional disorders induced by I/R, when compared with intact male mice or orchiectomized mice treated with dihydrotestosterone, a non-aromatizable isoform of testosterone. Thus, the protection afforded by orchiectomy is associated with increased expression of HSP-27, a heat shock protein important for maintenance of actin cytoskeletal integrity. These findings indicate that testosterone inhibits the heat shock response and may provide a new paradigm for design of therapies for I/R injury.