AbstractType VI secretion systems (T6SSs) can deliver diverse toxic effectors into eukaryotic and bacterial cells. Although much is known about the regulation and assembly of T6SS, the translocation mechanism of effectors into the periplasm and/or cytoplasm of target cells remains elusive. Here, we use theAgrobacterium tumefaciensDNase effector Tde1 to unravel the mechanism of translocation from attacker to prey. We demonstrate that Tde1 binds to its adaptor Tap1 through the N‐terminus, which harbors continuous copies of GxxxG motifs resembling the glycine zipper structure found in proteins involved in the membrane channel formation. Amino acid substitutions on G39xxxG43motif do not affect Tde1–Tap1 interaction and secretion but abolish its membrane permeability and translocation of its fluorescent fusion protein into prey cells. The data suggest that G39xxxG43governs the delivery of Tde1 into target cells by permeabilizing the cytoplasmic membrane. Considering the widespread presence of GxxxG motifs in bacterial effectors and pore‐forming toxins, we propose that glycine zipper‐mediated permeabilization is a conserved mechanism used by bacterial effectors for translocation across target cell membranes.