Sepsis causes mitochondrial oxidative injury and swelling. Ethyl pyruvate (EP) is a cytoprotective agent, while aquaporin-8 (AQP8) is a mitochondrial water channel that can induce mitochondrial swelling. We assessed whether EP protects mitochondria during sepsis, and whether AQP8 contributes to the underlying mechanisms. A cecal ligation and puncture (CLP) sepsis model was established in Sprague-Dawley rats, randomized to 3 groups: sham (n=20), CLP (n=59) and CLP+EP (n=51). All rats received postoperative intraperitoneal fluid resuscitation (30 ml/kg); the CLP+EP group also received intraperitoneal EP (100 mg/kg). Survival was assessed at 24 hours. Hepatic mitochondrial ultrastructure was characterized by electron microscopy. The membrane potential of isolated hepatic mitochondria was determined using JC-1 and flow cytometry. Mitochondrial AQP8 expression and cytochrome C (Cyt C) release were measured by Western blotting (values normalized to ß-actin). Survival in the sham, CLP and CLP+EP groups was 100%, 21% and 41%, respectively. Mitochondrial cross-sectional area was smaller in the CLP+EP group than in the CLP group (0.231±0.110 vs. 0.641±0.460 µm(2); P<0.001), with a tendency for a lower form factor (a measure of contour irregularity) in the CLP+EP group. Mitochondrial depolarization by CLP was inhibited by EP. Mitochondrial Cyt C release was higher in the CLP group than in the sham (1.211±0.24 vs. 0.48±0.03) or CLP+EP (0.35±0.39) groups. AQP8 expression was similar between groups, with a trend for lower expression in the CLP+EP group compared with the CLP group. EP improves sepsis outcome by targeting the mitochondrion, possibly through modulation of AQP8 expression.