Upper urinary tract obstruction is a common cause of renal dysfunction in children and adults. While there is clinical evidence of an increased male incidence and mortality rate with acute renal failure, the effect of gender and testosterone on obstructive renal injury has not previously been evaluated. We hypothesized that testosterone exacerbates pro-inflammatory TNF- production and pro-apoptotic and pro-fibrotic signaling during renal obstruction, resulting in increased apoptotic cell death and tubulointerstitial fibrosis. To study this, male, female, castrated male, and testosterone treated oophorectomized female rats were subjected to sham operation or 3 days of unilateral ureteral obstruction (UUO). Renal cortical tissue was then analyzed for tumor necrosis factor (TNF-) production, pro-apoptotic caspase 8, 9, and 3 activity, apoptotic cell death, pro-fibrotic transforming growth factor-1 (TGF-1) production, and -smooth muscle actin expression (-SMA). In a separate arm, glomerular filtration rate (GFR; inulin clearance) was measured in rats pre and post UUO. Male and testosterone treated oophorectomized female rats demonstrated a significant increase in TNF- production, caspase activity, apoptotic cell death, tubulointerstitial fibrosis, and renal dysfunction during UUO as compared to castrated males and normal female rats subjected to the same time course of obstruction. These results demonstrate that endogenous testosterone production in normal male rats and testosterone exogenously administered to oophorectomized females significantly increases TNF- production and pro-apoptotic and pro-fibrotic signaling during renal obstruction, resulting in increased apoptotic cell death, tubulointerstitial fibrosis, and renal dysfunction.