A mismatch between oxygen supply and metabolic tissue demand leads to the development of hypoxia, which contributes to hepatic dysfunction and liver injury during sepsis. Recent evidence shows that hepatic H2S levels are increased during sepsis and the inhibition of endogenous H2S production significantly improves survival in septic mice; however, the precise mechanism is not known. The present study was designed to investigate the effect of H2S on hepatic microcirculation and mitochondrial dynamics during sepsis. We hypothesized that H2S contributes to hepatic dysfunction during sepsis by potentiating microvascular dysfunction and alterations in mitochondrial dynamics. Using intravital microscopy, we show that portal infusion of H2S and ET-1 is associated with sinusoidal constriction and inhibition of endogenous H2S attenuates the sensitization of the sinusoids to the constrictor effect of ET-1 and that the response to ET-1 is sex-related. Moreover, we show that endotoxemia leads to alterations in mitochondrial function, which is also sex-related. We furthered our investigation in vitro where we show that endotoxemia results in mitochondrial depolarization, alterations in mitochondrial dynamics, and increases in stress fiber formation, which contribute to vascular permeability observed in sepsis. We conclude that the contribution of H2S to hepatic microcirculatory and mitochondrial dysfunction is responsible, at least in part, for its deleterious effects during sepsis.