Cytoplasmic membrane-bound connexin 43 (Cx43) proteins oligomerize into hexameric channels (hemichannels) that can sometimes dock with hemichannels on adjacent cells to form gap junctional (GJ) channels. However, the possible role of Cx43 hemichannels in sterile and infectious inflammatory diseases has not been adequately defined due to the lack of selective interventions. Here we report that a proinflammatory mediator, the serum amyloid A (SAA), resembled bacterial endotoxin by stimulating macrophages to up-regulate Cx43 expression and double-stranded RNA-activated protein kinase R (PKR) phosphorylation in a TLR4-dependent fashion. Two well-known Cx43 mimetic peptides, the GAP26 and TAT-GAP19, divergently affected macrophage hemichannel activities in vitro, and differentially altered the outcome of lethal sepsis in vivo. By screening a panel of Cx43 mimetic peptides, we discovered that one cysteine-containing peptide, P5 (ENVCYD), effectively attenuated hemichannel activities, and significantly suppressed endotoxin-induced release of ATP and HMGB1 in vitro. In vivo, the P5 peptide conferred a significant protection against hepatic ischemia/reperfusion injury and lethal microbial infection. Collectively, these findings have suggested a pathogenic role of Cx43 hemichannels in sterile injurious as well as infectious inflammatory diseases possibly through facilitating extracellular ATP efflux to trigger PKR phosphorylation/activation.