We previously demonstrated that renal peptidyl arginine deiminase-4 (PAD4) is induced after renal ischemia and reperfusion (IR) injury and exacerbates acute kidney injury (AKI) by increasing the renal tubular inflammatory response. Here, we tested whether genetic ablation of PAD4 attenuates renal injury and inflammation after IR in mice. After 30 min renal IR, PAD4 wild type mice develop severe AKI with large increases in plasma creatinine, neutrophil infiltration as well as significant renal tubular necrosis, apoptosis and pro-inflammatory cytokine generation. In contrast, PAD4 deficient mice are protected against ischemic AKI with reduced real tubular neutrophil infiltration, renal tubular necrosis and apoptosis. In addition, hepatic injury and inflammation observed in PAD4 wild type mice after renal IR is significantly attenuated in PAD4 deficient mice. We also show that increased renal tubular PAD4 expression after renal IR is associated with translocation of PAD4 from the nucleus to the cytosol. Consistent with PAD4 cytosolic translocation, we show increased renal tubular cytosolic peptidyl-citrullination after ischemic AKI. Mechanistically, recombinant PAD4 treatment increased nuclear translocation of NFkB in cultured human as well as murine proximal tubule cells that is inhibited by a PAD4 inhibitor (2-chloroamidine). Taken together, our studies further support the hypothesis that renal tubular PAD4 plays a critical role in renal IR injury by increasing the renal tubular inflammatory response and neutrophil infiltration after renal IR perhaps by interacting with the pro-inflammatory transcription factor NFkB in the cytosol and promoting its nuclear translocation.