Ancient, inactive copies of transposable elements of the PIF/Harbinger superfamily have been described in vertebrates. We reconstructed components of the Harbinger3_DR transposon in zebrafish, including a transposase and a second, transposon-encoded protein that has a Myb-like trihelix domain. The reconstructed Harbinger transposon shows efficient cut-and-paste transposition in human cells and preferentially inserts into a 15-bp consensus target sequence. The Myb-like protein is required for transposition and physically interacts with the N-terminal region of the transposase via its C-terminal domain. The Myb-like protein enables transposition in part by promoting nuclear import of the transposase, by directly binding to subterminal regions of the transposon, and by recruiting the transposase to the transposon ends. We investigated the functions of two transposon-derived human proteins: HARBI1, a domesticated transposase-derived protein, and NAIF1, which contains a trihelix motif similar to that described in the Myb-like protein. Physical interaction, subcellular localization, and DNA-binding activities of HARBI1 and NAIF1 suggest strong functional homologies between the Harbinger3_DR system and their related, host-encoded counterparts. The Harbinger transposon will serve as a useful experimental system for transposon biology and for investigating the enzymatic functions of domesticated, transposon-derived cellular genes.