A possible application of acupuncture in alleviating depression-like behavioral changes and regulating serotonin signaling in the prefrontal cortex (PFC) of maternally-separated rat pups was investigated in this study. On postnatal day 15, rat pups were maternally-separated and received acupuncture stimulation at acupoint HT7 or ST36 once a day for 7 days. On postnatal day 21, the tail suspension test was performed and the PFC was harvested. Tissue levels of serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) were then measured by high-performance liquid chromatography and expression of serotonin transporter (5-HTT) and brain-derived neurotrophic factor (BDNF) were assessed by western blotting. Levels of 5-HT and 5-HIAA were not significantly changed, but the 5-HIAA/5-HT ratio was significantly increased by maternal separation. The immobility time of maternally-separated rat pups was increased, and increased 5-HTT expression and reduced BDNF level were observed in the PFC. But acupuncture stimulation at HT7 alleviated the behavioral change and regulated the changes of 5-HIAA/5-HT ratio, 5-HTT, and BDNF. In conclusion, acupuncture stimulation at HT7 can relieve maternal separation-induced changes, and we propose that regulation of the 5-HIAA/5-HT ratio and of 5-HTT expression by acupuncture stimulation are important acupuncture-induced benefits in this animal model of depression.