Endotoxemia, the systemic inflammatory host response to infection, leads to severe septic shock and multiple organ injury and dysfunction syndrome (MOPS), which cause mortality. Apolipoprotein A-I-Milano (apoAI(M)), a naturally occurring cysteine mutant of apoAI with dimers as its effective form, showed an enhanced cardiovascular protective activity compared with wild-type apoAI (apoAIwt). To investigate the role of recombinant high-density lipoprotein (rHDL) reconstituted with apoAI(M) (rHDL(M)) on endotoxemia and MOPS, we examined the anti-inflammatory, anti-oxidant, and protective effects of this cysteine mutant against organ injury in endotoxin-challenged rat models compared with rHDLwt. In the present study, we demonstrated for the first time that pretreatment with rHDL(M) significantly attenuated liver and renal dysfunction and histopathological features of lung injury in endotoxin-challenged endotoxemia rats. Administration of rHDL(M) to endotoxemia rats dramatically suppressed proinflammatory cytokines and adhesion molecule increase in tumor necrosis factor alpha, interleukin 1 beta, interleukin 6, and intercellular adhesion molecule 1. In addition, rHDL(M) pretreatment inhibited lipid peroxidation and enhanced total antioxidant capacity in vivo. In comparison with rHDLwt, rHDL(M) showed enhanced capacity on anti-inflammatory and anti-oxidant functions. In summary, administration of rHDL(M) protected endotoxin-challenged endotoxemia and MOPS through enhanced anti-inflammatory and anti-oxidant properties.