Cre/loxP-mediated DNA excision in germ cell lineages could contribute substantially to the study of germ cell biology in salmonids, which are emerging as a model species in this field. However, a cell type-specific Cre/loxP system has not been successfully developed for any salmonid species. Therefore, we examined the feasibility of Cre/loxP-mediated, germ cell-specific gene excision and transgene activation in rainbow trout. Double-transgenic (wTg) progeny were obtained by mating a transgenic male carrying cre with a transgenic female carrying the hsc-LRLG gene; cre was driven by rainbow trout vasa regulatory regions and the hsc-LRLG gene was made up of the rainbow trout heat-shock-cognate71 promoter, the DsRed gene flanked by two loxP sites, and the Egfp gene. PCR analysis, fluorescence imaging, and histological analysis revealed that excision of the loxP-flanked sequence and activation of Egfp occurred only in germ cells of wTg fish. However, progeny tests revealed that the excision efficiency of loxP-flanked sequence in germ cells was low (≤3.27%). In contrast, the other wTg fish derived from two different cre-transgenic males frequently excised the loxP-flanked sequence in germ cells (≤89.25%). Thus, we showed for the first time successful germ cell-specific transgene manipulation via the Cre/loxP system in rainbow trout. We anticipate that this technology will be suitable for studies of cell function through cell targeting, cell-linage tracing, and generating cell type-specific conditional gene knockouts and separately for developing sterile rainbow trout in aquaculture.