The medial preoptic nucleus (MPN) is a regulatory center for male sexual behavior. It consists of sexually dimorphic structures that are male biased, and these structures are found in the central part of the MPN (MPNc). The bed nucleus of the stria terminalis (BNST) also participates in male sexual behavior, and receives efferent neural projections from the MPNc. In this study, we examined if MPNc neurons projecting to the BNST are activated in male rats displaying sexual behavior. Fluoro-Gold (FG; a retrograde neural tracer) was injected into the BNST of male rats, which were separated into two groups: (1) those in contact with estrous female rats and displayed sexual behavior followed by ejaculation and (2) those without contact with estrous female rats. In both groups, protein expression of c-Fos (a neuronal activity marker) and calbindin (a location marker of the MPNc) were detected by fluorescent immunohistochemistry. The number of c-Fos-immunoreactive cells with or without FG labeling in the MPNc was also measured. The number of c-Fos-immunoreactive cells significantly increased following ejaculation. Approximately 10% of FG-labeled cells in ejaculation male rats were immunoreactive for c-Fos, and this percentage value was significantly higher in this group compared with control male rats. Overall, these results suggest that efferent projections from the MPNc to the BNST function to control sexual behavior in male rats.