The present study investigated the possible role of acupuncture in alleviating depression-like behavioral changes and examined changes in the levels of serotonin (5-HT), dopamine (DA), and their metabolites in the hippocampus (HP) and prefrontal cortex (PFC) of maternally separated rat pups. On postnatal day 15, rat pups were maternally separated and received acupuncture stimulation at acupoint HT7 or ST36 once a day for 7 days. Then, on postnatal day 21, a tail suspension test was performed, and the HP and PFC were harvested. Levels of 5-HT, 5-hydroxyindole-3-acetic acid (5-HIAA), DA, and 3,4-dihydroxyphenylacetic acid (DOPAC) in the tissue and corticosterone (CORT) in plasma were then measured. The total duration of immobility in maternally separated rat pups increased after maternal separation, and this increase was alleviated by acupuncture stimulation at HT7. The 5-HIAA/5-HT ratio and the levels of 5-HT and 5-HIAA were not significantly changed, but those of the DA and the DOPAC/DA ratio were significantly lower and that of CORT was significantly higher after maternal separation. The maternal separation-induced changes of the DOPAC/DA ratio and the CORT level significantly alleviated after acupuncture stimulation at HT7. These results suppose that the functional recovery of prefrontal-limbic system by acupuncture stimulation plays an important role in acupuncture-induced benefits in this animal model of depression.