Recent evidence indicates that high-fat diet (HFD) induces alterations in gut microbiota and barrier function, which are linked to hepatic and intestinal inflammation that promote metabolic syndrome. Similarly, antibiotic-induced dysbiosis influences host metabolism leading to fat accumulation. Melatonin, known as the modulator of host’s circadian clock, was also suggested to regulate circadian rhythm of gut bacteria1. Aim of the study was to elucidate the role of exogenous melatonin in the changed oxidative status of the liver and intestine by short-term ingestion of HFD in the absence or presence of antibiotic intake. Six-week-old male Sprague-Dawley rats (n= 48) were pair-fed with chow or HFD (45% fat), and were given either tap water or melatonin (4 mg/kg/day) or melatonin+antibiotics (MA, neomycin+ampicillin+metronidazole; 1 g/L each) in drinking water for two weeks. Faecal output was used as an index for colonic motility and intestinal transit was assessed using charcoal propagation. Trunk blood was used for measurement of plasma triglyceride, HDL, LDL and glucose levels. Liver, epididymal and perirenal fat were weighed. Myeloperoxidase activity (MPO), malondialdehyde (MDA), glutathione (GSH), nitric oxide (NO), chemiluminescence (CL) levels were measured in liver and ileum, and ileal villus height was determined histologically. Bacterial colonies were counted in faeces. Statistical analysis was made by ANOVA and Student’s t tests. HFD increased perirenal fat weight (p<0.05), but melatonin prevented fat accumulation. In MA treated HFD group, increases in perirenal fat and plasma glucose were abolished, while triglyceride levels were further elevated (p<0.05). Reduced colonic and intestinal transits in HFD were further delayed with melatonin, while addition of antibiotics to melatonin abolished HFD-induced delay in intestinal transit (p<0.001). Villus heights were reduced significantly in HFD-rats with MA treatment (p<0.01). Hepatic and ileal NO and CL levels that were elevated by HFD were lower in melatonin-and MA-treated rats. Ingestion of HFD increased ileal and hepatic GSH content without changing MPO and MDA levels, while MA combination depleted GSH (p<0.001). Either melatonin (10-folds) or HFD (7-folds) elevated total bacterial count, while antibiotics-induced diminution in faecal bacteria was partially prevented by melatonin in rats fed with either diet. Two-week HFD delayed gut motility and induced a low-level inflammation with a compensatory GSH increment. Melatonin alleviated inflammatory response and fat accumulation, but further delayed motility. Reversal of GSH and motility responses by antibiotic-induced dysbiosis suggest that melatonin-modulated gut microbiota are involved in altered GSH metabolism and gastrointestinal motility due to acute exposition to pro-oxidant lipids.