Hydrogen sulphide is an endogenous inflammatory mediator produced by cystathionine-γ-lyase (CSE) in macrophages. To determine the role of H2S and macrophages in sepsis, we used small interference RNA (siRNA) to target the CSE gene and investigated its effect in a mouse model of sepsis. Cecal ligation puncture (CLP)-induced sepsis is characterized by increased levels of myeloperoxidase (MPO) activity, morphological changes in liver and pro-inflammatory cytokines and chemokines in the liver and lung. SiRNA treatment attenuated inflammation in the liver and lungs of mice following CLP-induced sepsis. Liver MPO activity increased in CLP-induced sepsis and treatment with siRNA significantly reduced this. Similarly, lung MPO activity increased following induction of sepsis with CLP while siRNA treatment significantly reduced MPO activity. Liver and lung cytokine and chemokine levels in CLP-induced sepsis reduced following treatment with siRNA. These findings show a crucial pro-inflammatory role for H2S synthesized by CSE in macrophages in sepsis and suggest CSE gene silencing with siRNA as a potential therapeutic approach for this condition.