In recent decades, several mass mortalities were recorded in riverine ayu (Plecoglossus altivelis) in Tokyo Metropolis, Hiroshima Prefecture, and Yamaguchi Prefecture, Japan; in these outbreaks, microbiological and pathological examinations revealed Edwardsiella ictaluri as the causative agent. In this study, histopathological findings and immunohistochemical localization of the bacteria following experimental infection of ayu were discussed. Infection experiments were performed using 44 healthy cultured ayu fingerlings using E. ictaluri isolate (H90). The fish were injected with the isolate intraperitoneally with a dose of 5.1 × 105 cfu/fish, while the control fish were injected with sterile phosphate buffered saline. The fish were observed for clinical signs, with daily collection of dead fish, and isolation of bacteria from the posterior kidney was performed and confirmed to be E. ictaluri by slide agglutination using anti‐PH0744 serum. Daily collection of five moribund fish for necropsy and tissue specimens collection from hepatopancreas, spleen, posterior kidney, gills, brain, heart, and intestine for histopathological and immunohistochemical examination. Post‐mortem lesions were recorded as exophthalmia, bloody ascitis, hemorrhagic kidney and distended gallbladder, meningio‐encephalitis, hemorrhagic vent, and petechial hemorrhages on viscera. Histopathological examination revealed diffuse severe congestion in blood vessels and several degenerative and necrotic changes inconcurrent with positive antigenic staining by immunohistochemistry.