Sepsis, a life-threatening disease with high morbidity and mortality in critically ill patients, usually leads to serious complications including liver damage and dysregulated metabolic homoeostasis. The aim of this study was to evaluate the therapeutic potential of melatonin in rats with caecal ligation and puncture (CLP)-induced sepsis, which mimics critical infections in humans and explore the underlying molecular mechanisms. Male Sprague-Dawley rats received CLP surgery under anaesthesia to induce polymicrobial sepsis. Melatonin (20 mg/kg) was intraperitoneally (i.p.) injected every 12 h for 7 days after CLP, with or without intraperitoneal injection of the SIRT1 inhibitor EX527 (5 mg/kg). Markers of glucose metabolism, inflammation, liver function and associated signaling pathway were measured. Septic rats exhibited marked inhibition of the hepatic SIRT1/STAT3 pathway, along with increased blood glucose levels and hepatic gluconeogenesis. Melatonin administration efficiently attenuated liver dysfunction and glucose metabolism disorders by promoting hepatic SIRT1 expression and STAT3 phosphorylation. Furthermore, inhibition of SIRT1 by EX527 significantly diminished the protective effects of melatonin on sepsis induced liver injury, hyperglycaemia and STAT3 inactivation. These results emphasize that melatonin is a potential therapeutic agent for sepsis-associated liver injury and glucose metabolism disorders, possibly acting by targeting SIRT1-mediated STAT3 activation in the liver.