In an animal model of post-traumatic stress disorder (PTSD), our previous studies showed mitochondrial stress-induced apoptosis in the hippocampus. Metformin, the most commonly prescribed anti-diabetic drug, exerts its effects through 5'-adenosine monophosphate-activated protein kinase (AMPK) activation. It was shown that a neuroprotective role was gradually established against stroke, spinal cord injury and Parkinson's disease. The aim of this study was to explore the role of the AMPK pathway in neuronal apoptosis in the hippocampus using a rat model of PTSD. The model PTSD rats received acute exposure to prolonged stress (single prolonged stress, SPS), followed by examination of the effects of genes and/or proteins related to the AMPK and oxidative stress pathways in the hippocampus with or without metformin preconditioning. The results indicated that the level of phosphorylated AMPK was markedly increased after SPS. Metformin protected the hippocampus as evidenced by abolishing down-regulation of the AMPK pathway and up-regulating expression of oxidative stress-related genes. These results indicated that metformin attenuated oxidative stress in the hippocampus in rats under SPS. AMPK pathway activation may be a novel therapeutic protocol for PTSD patients.