We have previously shown that PACAP in the ventromedial hypothalamic nucleus (VMH) enhances feeding at night and after fasting, but inhibits feeding during daytime. On the other hand, the neuropeptide galanin is also shown to be highly expressed in the hypothalamus and involved in feeding regulation. In this study, we investigated the possible involvement of VMH-PACAP in the dorsomedial hypothalamic nucleus (DMH)-galanin signaling for mouse feeding behavior. The expression of galanin in the hypothalamus was significantly increased by fasting, but this increase was cancelled in PACAP knockout mice. Furthermore, overexpression of PACAP in the VMH increased the expression of galanin, while knockdown of PACAP in the VMH decreased the expression of galanin, indicating that the expression of galanin in the hypothalamus might be regulated by PACAP in the VMH. Therefore, we expressed synaptophysin-EGFP chimeric protein in PACAP neurons in the VMH, and visualized the neural projections to the hypothalamic region where galanin was highly expressed. Strong EGFP signal was observed in the DMH, suggesting that PACAP-expressing neurons in the VMH projected to the DMH. Furthermore, in the DMH, immunostaining of galanin showed that galanin expression increased with fasting, but this was not observed in PACAP knockout mice. When galanin in the DMH was knocked down by shRNA treatment, food intake at night and after fasting was decreased, whereas food intake during daytime was increased, as shown in the PACAP knockout mice. These results suggested that VMH-PACAP may regulate mouse feeding behavior through DMH-galanin.