A major impediment to the commercialization and cultivation of transgenic fish is the potential ecological risks they pose to natural environments: a problem that could be solved by the production of sterile transgenic fish. Here, we have developed an on-off reproductive containment strategy for fish that renders the offspring sterile but leaves their parents fertile. TG1 (Tol2-CMV-GFP-pA-CMV-gal4-pA-Tol2) and TG2 (Tol2-CMV-RFP-pA-5 × UAS-as/dnd-pA-Tol2) zebrafish lines were established using a GAL4/UAS system. While the parental lines remained fertile, in the hybrid offspring, GAL4 induced 5 × UAS to drive the transcription of antisense dnd, which significantly down-regulated endogenous dnd expression. This disrupted the migration of primordial germ cells (PGCs), led to their apoptosis, and resulted in few or no PGCs migrating to the genital ridge. This process induced sterility or reduced fertility in adult fish. This on-off strategy is a potentially effective means of generating sterile fish for commercialization while retaining fertility in brood stocks, and offers a novel method to mitigate the ecological risks of fish introductions.