The Tol2 transposon system is a useful gene transduction technique, but the injection of mRNA is not sufficiently effective in Xenopus embryos to express Tol2 transposase (Tol2TP). To overcome this, we bacterially synthesized recombinant Tol2TP (rTol2TP) protein and showed that rTol2TP efficiently excised the Tol2 element from an injected donor plasmid in Xenopus embryos. Furthermore, injected embryos exhibited uniform and ubiquitous expression of an EGFP reporter gene placed within the Tol2 element. Importantly, size-exclusion chromatography suggests that rTol2TP forms a tetramer, which differs from the reported hexamer formed by Hermes transposase, although both belong to the same hAT family. The use of rTol2TP may facilitate efficient gene transduction in Xenopus, and the biochemical characterization of Tol2TP.