Bacillus thuringiensis (Bt) Cry1Ac protein is a toxin against different leaf-eating lepidopteran insects that attack poplar trees. In the present study, the mode of migration of the Bt-Cry1Ac protein within poplar grafts was investigated. Grafting was done using Pb29 (transgenic poplar 741 with cry1Ac genes), CC71 (transgenic poplar 741 with cry3A genes), non-transgenic poplar 741 and non-transgenic Populus tomentosa, either as scion or as rootstock. In order to detect migration of Bt-Cry1Ac protein from one portion of the graft union to different tissues in the grafted plant, ELISA analysis was employed to assess the content of Bt-Cry1Ac protein in the phloem, xylem, pith and leaves of the grafted poplar. To further verify migration of Bt-Cry1Ac protein, Clostera anachoreta larvae, which are susceptible to Bt-Cry1Ac protein, were fed leaves from the control graft (i.e., graft portion that originally did not contain Bt-Cry1Ac protein). The results showed that Bt-Cry1Ac protein was transported between rootstock and scion mainly through the phloem. Migration of Bt-Cry1Ac protein in the grafted union was also evidenced in that the leaves of the control graft did have a lethal effect on C. anachoreta larvae in laboratory feeding experiments.