Tol1 is a DNA-based transposable element first identified from an albino mutant medaka fish. It has been demonstrated to function as an efficient gene transfer vector in mammalian cells. We now demonstrate Tol1 germline transgenesis in zebrafish. A construct containing the green fluorescence protein (GFP) reporter gene inserted between the Tol1 arms was microinjected together with Tol1 transposase mRNA into fertilized eggs. Sustained GFP expression was observed in 88% of 1-month-old fish, suggesting efficient transposon integration into somatic cells. Eleven of 24 adult GFP-positive fish yielded GFP-positive progeny. Sequencing analysis of Tol1 insertion sites in GFP-positive progeny confirmed Tol1 transposition-mediated integrations into zebrafish chromosomes. We also observed functional independence of the Tol1 transposase-substrate system from that of Tol2, another medaka-derived transposon. Coupled with its previously demonstrated maximal cargo capacity of >20 kb, Tol1 could serve as a useful addition to the zebrafish genetic engineering toolbox.