Rhodococcus equi was isolated from the granulomatous lesions of the lung, kidney, liver, and hepatic, mesenteric, and abomasum lymph nodes of a Japanese black heifer. R. equi isolates were analyzed by polymerase chain reaction for virulence-associated protein genes. The vapN gene was detected in all the isolates examined. This is the first report in which vapN-positive R. equi was isolated from cattle in Japan.