The calcium sensitizer levosimendan is used in treatment of decompensated heart failure and may also exhibit anti-inflammatory properties. We examined whether treatment with levosimendan is substantially beneficial in mice with cecal ligation and puncture-induced polymicrobial sepsis, and its arbitration mechanism was explored in the mouse macrophage cell line RAW264.7.,Laboratory and animal/cell research.,University research laboratory.,BALB/c mice (8-10 wk old) and mouse macrophage cell line RAW264.7 cells.,Levosimendan (0.5 g/kg/min) was administered to mice through an osmotic pump that was implanted into the peritoneal cavity immediately following surgery. In RAW264.7 cells, levosimendan was added to the culture medium 30 minutes before lipopolysaccharide.,When levosimendan was continuously administered to cecal ligation and puncture-induced septic mice, a significant improvement of left ventricular function was found without any change in heart rate, and hypotension was significantly mitigated. Furthermore, levosimendan conferred substantial protection against sepsis-associated inflammation in mice, as indicated by reduced lung injury and decreased blood proinflammatory and chemotactic cytokine levels. These beneficial effects of levosimendan led to a significant improvement of survival in mice after cecal ligation and puncture. In endotoxin-stimulated RAW264.7 macrophages, treatment with levosimendan and pimobendan suppressed overproduction of proinflammatory and chemotactic cytokines. Levosimendan and pimobendan were without effect on activation of the nuclear factor-B, mitogen-activated protein kinase, and Akt pathways. Instead, levosimendan and pimobendan prevented high mobility group box 1 release from the nucleus to the extracellular space in macrophages. This was associated with inhibition of the Rho kinase signaling pathway. The elevated serum high mobility group box 1 levels in cecal ligation and puncture-induced septic mice were also inhibited by continued administration of levosimendan and pimobendan.,We define a novel mechanism for the anti-inflammatory action of levosimendan and suggest that the pharmacological profiles of levosimendan as both an inotrope and an anti-inflammatory agent could contribute to its clinical benefit in patients with sepsis with heart problems.