Pseudotuberculosis caused by Photobacterium damselae subsp. piscicida is a serious problem in fish culture industry. The initial stage of host-pathogen interaction is primarily important to understand infectious disease. However, the mode of transmission and route of infection of the agent are not fully studied. In this study, changes in the distribution of the bacterium on and in yellowtail Seriola quinqueradiata body during infection process was investigated using immersion challenge. The result revealed that external regions such as the lower jaw, pectoral fin, skin and gills permitted bacterial adherence soon after immersion. Viable bacterial counts increased in the gills soon after infection, then they increased in the blood, spleen and kidney. Moreover, fish were exposed to bacteria at the gills or posterior part of body surface to elucidate the primary infection route. The fish group challenged at gills showed higher mortality than the other group. These results reveal that the gills are the primary regions for P. damselae subsp. piscicida to invade and proliferate in yellowtail.