Prostacyclin and peroxisome proliferator-activated receptors (PPAR) protect against ischemia-reperfusion (I/R) injury by the induction of an anti-inflammatory pathway. In this study, we examined the prostacyclin-enhanced protective effect of PPARalpha in I/R-induced kidney injury. PPAR-alpha reduced the NF-kappaB-induced overexpression of TNF-alpha and apoptosis in cultured kidney cells. In a murine model, pretreating wild-type (WT) mice with a PPAR-alpha activator, docosahexaenoic acid (DHA), significantly reduced I/R-induced renal dysfunction (lowered serum creatinine and urea nitrogen levels), apoptotic responses (decreased apoptotic cell number and caspase-3, -8 activation), and NF-kappaB activation. By comparison, I/R-induced injury was exacerbated in PPAR-alpha knockout mice. This indicated that PPAR-alpha attenuated renal I/R injury via NF-kappaB-induced TNF-alpha overexpression. Overexpression of prostacyclin using an adenovirus could also induce PPAR-alpha translocation from the cytosol into the nucleus to inhibit caspase-3 activation. This prostacyclin/PPAR-alpha pathway attenuated TNF-alpha promoter activity by binding to NF-kappaB. Using a cAMP inhibitor (CAY10441) and a prostacyclin receptor antibody, we also found that there was another prostacyclin/IP receptor/cAMP pathway that could inhibit TNF-alpha production. Taken together, our results demonstrate for the first time that prostacyclin induces the translocation of PPAR-alpha from the cytosol into the nucleus and attenuates NF-kappaB-induced TNF-alpha activation following renal I/R injury. Treatments that can augment prostacyclin, PPAR-alpha, or the associated signaling pathways may ameliorate conditions associated with renal I/R injury.