Acupuncture has been shown to have therapeutic effects on many mental disorders such as depression, anxiety, and substance abuse in human and animal studies. Maternal separation (MS) is a risk factor in the development of mood-related disorders such as depression. This study investigated the effects of acupuncture during MS by evaluating locomotion as behavioral markers of depression and by observing changes in c-Fos expression in the limbic system (hippocampus, amygdala and cingulate cortex) to elucidate the mechanism of the therapeutic effect. The employed acupoint, Shinmun (HT7), has been clinically used to treat mental disorders. MS for 7 days beginning on postnatal day 14 induced a significant decrease in locomotion, while acupuncture treatment at acupoint HT7 resulted in a significant increase in locomotion. c-Fos immunoreactive cells in area dentate gyrus (DG) of the hippocampus were increased in the MS group, but significantly decreased in the acupuncture group. These findings suggest that acupuncture has an effect on the depression-like disorder caused by MS, possibly by modulating c-Fos expression in DC of the hippocampus.