Upon intravenous injection of tumour necrosis factor (TNF) in mice, a systemic inflammatory response syndrome (SIRS) is initiated, characterized by an acute cytokine storm and induction of vascular hyperpermeability. Connexin43 hemichannels have been implicated in various pathological conditions, e.g. ischemia and inflammation, and can lead to detrimental cellular outcomes. Here, we explored whether targeting connexin43 hemichannels could alleviate TNF-induced endothelial barrier dysfunction and lethality in SIRS. Therefore, we verified whether administration of connexin43-targeting-peptides affected survival, body temperature and vascular permeability in vivo. In vitro, TNF-effects on connexin43 hemichannel function were investigated by single-channel studies and Ca2+-imaging. Blocking connexin43 hemichannels with TAT-Gap19 protected mice against TNF-induced mortality, hypothermia and vascular leakage, while enhancing connexin43 hemichannel function with TAT-CT9 provoked opposite sensitizing effects. In vitro patch-clamp studies revealed that TNF acutely activated connexin43 hemichannel opening in endothelial cells, which was promoted by CT9, and inhibited by Gap19 and intracellular Ca2+-buffering. In vivo experiments aimed at buffering intracellular Ca2+, and pharmacologically targeting Ca2+/calmodulin-dependent protein kinase-II, a known modulator of endothelial barrier integrity, demonstrated their involvement in permeability alterations. Our results demonstrate significant benefits of inhibiting connexin43 hemichannels to counteract TNF-induced SIRS-associated vascular permeability and lethality.