Acute liver injury is a clinical hallmark of endotoxemia regarding the features of septic organ failure. In this process, interleukin (IL)-6 and IL-10 are key contributors for eliciting pro- and anti-inflammatory responses, respectively. In contrast, heme oxygenase-1 (HO-1) provides a defense mechanism against endotoxemia by controlling the IL-6/IL-10 balance, but how higher levels of HO-1 are sustained under pathological conditions remains unknown. Using a mouse model of endotoxemia, we provide evidence to show that hepatocyte growth factor (HGF) enhances HO-1 expression in macrophages, thereby up-regulating IL-10 and down-regulating IL-6 productions. Lipopolysaccharide (LPS)-treated mice manifested acute liver injury similar to that observed in septic patients, while administration of recombinant HGF enhanced expression of HO-1 by hepatic macrophages in vivo. As a result, HGF blocked the onset of hepatic injuries in LPS-treated mice. More importantly, when an HO-1 inhibitor (Sn-PP) was administered with HGF into LPS-treated mice, the protective effects of HGF against hepatic injury were attenuated. Furthermore, Sn-PP partially restored the HGF-mediated decrease in plasma IL-6 levels, while it inhibited the HGF-stimulated increase in plasma IL-10 levels. In the culture of macrophages (Raw264.7), HGF enhanced the LPS-mediated HO-1 induction, and this effect was abolished by cycloheximide, but not by actinomycin-D, thus suggesting that a post-transcriptional pathway is involved in HGF-mediated up-regulation of HO-1. Based on the current data, we conclude that up-regulation of HO-1 plays an important role in HGF-mediated hepatoprotection during endotoxemia, by favoring production of IL-10 over IL-6.