The medial preoptic area, which plays an essential role in the control of sexual behavior in rats, contains a sexually dimorphic nucleus that consists of neurons expressing calbindin-D28 K (Calb) that is referred to as the CALB-SDN. The CALB-SDN is larger and contains more Calb neurons in males than in females. The physiological functions of the CALB-SDN are not fully understood; however, CALB-SDN neurons are activated during sexual behavior in males, suggesting that the male CALB-SDN is involved in regulation of sexual behavior. However, no information exists about the physiological functions of the female CALB-SDN. In the present study, we performed an immunohistochemical analysis of c-Fos, a neuronal activity marker, in the CALB-SDN of female and male rats that had copulated with conspecifics of the opposite sex to determine whether neurons of the female CALB-SDN are activated during copulation and whether the neuronal activity of the CALB-SDN differs between sexes. The numbers of c-Fos-immunoreactive cells with or without Calb-immunoreactivity (c-Fos+/Calb+ and c-Fos+/Calb− cells) were greater in the CALB-SDN of rats that had copulated than in rats that had not copulated in each sex. Although the number of Calb+ cells in the CALB-SDN was smaller in females than in males, the increase in the number of c-Fos+/Calb+ cells in the female CALB-SDN with copulation was comparable to that in the male CALB-SDN with copulation. The increase in the number of c-Fos+/Calb− cells in the CALB-SDN with copulation was more prominent in males than in females. These results suggest that CALB-SDN neurons are activated during copulation in both sexes. The patterns of neuronal activation in the CALB-SDN during copulation may differ between sexes.