Epidemiological studies have demonstrated that asymptomatic infection of Borna disease virus (BDV) is found in various species of animals in Japan. Recent reports have also revealed that neurological diseases caused by this virus could exist in horses, cattle, a dog, and cats in this country. In this study, we investigated seroprevalence of BDV antibodies in Japanese black cows reared in Kyushu, the southernmost main island of Japan, using ELISA and Western-immunoblotting. Of 101 serum samples, 11 (10.9%) and 21(20.7%) sera were identified as having antibodies to the BDV N and P antigens, respectively. Among the positive sera, three cows (2.9%) were seropositive for both of the antigens. Furthermore, interestingly, only female cows showed antibodies to P, whereas N antibodies were detected in male and female cows with a comparative ratio. Together with previous studies, our results indicate that BDV might be widely spread in cattle raised in Japan. Furthermore, this is the first report to show that beef cattle, Japanese black cattle, have antibodies against a possible zoonotic pathogen, BDV.