Kallistatin, a plasma protein, has been shown to exert multi-factorial functions including inhibition of inflammation, oxidative stress and apoptosis in animal models and cultured cells. Kallistatin levels are reduced in patients with sepsis and in lipopolysaccharide (LPS)-induced septic mice. Moreover, transgenic mice expressing kallistatin are more resistant to LPS-induced mortality. Here, we investigated the effects of human kallistatin on organ injury and survival in a mouse model of polymicrobial sepsis. In this study, mice were injected intravenously with recombinant kallistatin (KS3, 3 mg/kg; or KS10, 10 mg/kg body weight) and then rendered septic by caecal ligation and puncture 30 min later. Kallistatin administration resulted in a > 10-fold reduction of peritoneal bacterial counts, and significantly decreased serum tumour necrosis factor-α, interleukin-6 and high mobility group box-1 (HMGB1) levels. Kallistatin also inhibited HMGB1 and toll-like receptor-4 gene expression in the lung and kidney. Administration of kallistatin attenuated renal damage and decreased blood urea nitrogen and serum creatinine levels, but increased endothelial nitric oxide synthase and nitric oxide levels in the kidney. In cultured endothelial cells, human kallistatin via its heparin-binding site inhibited HMGB1-induced nuclear factor-κB activation and inflammatory gene expression. Moreover, kallistatin significantly reduced apoptosis and caspase-3 activity in the spleen. Furthermore, kallistatin treatment markedly improved the survival of septic mice by 23% (KS3) and 41% (KS10). These results indicate that kallistatin is a unique protecting agent in sepsis-induced organ damage and mortality by inhibiting inflammation and apoptosis, as well as enhancing bacterial clearance in a mouse model of polymicrobial sepsis.