The life cycle of the fish ectoparasitic isopod Gnathia trimaculata is described based on both field samplings and laboratory observations. Species identification of the larvae was based on morphological observation and supported by molecular analysis. As the results of field samplings in several sites of southwestern and central Japan (24-34[degrees]N, 124-139[degrees]E) from 2005-2011, approximately 900 third-stage larvae of G. trimaculata were found on 25 elasmobranch species, and 220 first- and second-stage larvae were found on three teleost species. No third-stage larvae were found on the teleosts, and the larvae of younger stages never infested elasmobranchs. Therefore, G. trimaculata is supposed to shift its host from teleosts to elasmobranchs as it develops. We discuss the developmental periods, life span, distribution, and predation risk of the present species compared with other gnathiid species.