Maternal separation (MS) is a risk factor in the development of mood-related disorders such as depression. Human and animal studies support the involvement of neuropeptide Y (NPY) in the pathology of depression. To investigate the effect of acupuncture on depression-like behavior and examine changes in NPY expression associated with MS, we observed body weight and locomotor activity, and performed NPY immunohistochemistry in the hippocampus. MS for 7 days beginning on postnatal day 14 induced a significant decrease in body weight and locomotion, while acupuncture treatment at acupoint Shenmen (HT7) resulted in a significant increase in both. NPY-immunoreactive cells in area CA1 and the dentate gyrus were decreased in the MS group, but significantly increased in the acupuncture group. These findings suggest that acupuncture has an effect on the depression-like disorder caused by MS, possibly by modulating NPY expression in the hippocampus.