Akabane virus (AKAV) is an arbovirus belonging to the family Bunyaviridae, genus Orthobunyavirus. AKAV consists of three-segment (L, M, and S RNA segments), negative single-stranded RNA. The aim of this study was to investigate an in situ hybridization method (ISH) in a Vero E6 cell line infected with Akabane virus. The 320 base pair amplicon was obtained by RT-PCR with a primer pair and labeled with digoxigenin. Akabane virus RNAs were seen as a granular pattern in the cytoplasm of infected cells. As a result, the expression of the particular Akabane virus gene area was successfully disclosed in the current investigation using the ISH method with a digoxigenin-labeled probe.