Abstract: Primordial germ cells (PGCs) give rise to gametes which transmit the genetic information to next generation, therefore PGCs provide us an ideal cell type for genetic manipulation. Homologous recombination (HR) is the most efficient technique to create designed genetic modifications, however, its efficiency is rather low in vertebrates. In this study, by using zebrafish as an in vivo model, we aimed to enhance the efficiency of HR in zebrafish PGCs. First, we injected UAS:mRFP-nos1 construct into Tg (kop:KalTA4) embryos to label the transgenic PGCs, and we showed that screening of PGCs-specific mRFP expression led to relatively high-efficient germline transmission of transgene. Then we established an in vivo assay to evaluate the HR frequency in PGCs. We further revealed that suppression of the activities of DNA ligase IV (Lig4) and Xrcc6 (previously known as Ku70) could significantly increase the HR efficiency, not only at whole embryo level but also in PGCs. We proposed that the Tg(kop:KalTA4) line could be used as an effective platform for HR-mediated gene targeting.