We have previously shown that transduction of HOXD3, one of homeobox genes, into human lung cancer A549 cells enhances cell motility, invasion and metastasis. In the present study, we examined the roles of integrin β3 which was up-regulated by HOXD3-overexpression in the HOXD3-induced motility of A549 cells. We first established integrin β3-transfectants and compared their motile activity to those of the HOXD3-transfected, control-transfected and parental cells by three different assays.The integrin β3-transfectants as well as the HOXD3-transfectants formed heterodimer with integrin αv subunit, and showed highly motile activities assessed by haptotaxis or phagokinetic track assay compared to the control transfectants or parental cells. In vitro wound-healing assay revealed that migratory activities were graded as the HOXD3-transfectants > the integrin β3-transfectants > the control transfectants or parental cells. E-cadherin was expressed in the integrin β3-transfectants but not expressed in the HOXD3-transfectants. An addition of function-blocking antibody to E-cadherin into the wound-healing assay promoted the migratory activity of the integrin β3-transfectants, suggesting that E-cadherin prevented the cells from dissociating from the wound edges. These results indicate that increased expression of integrin αv β3 and loss of E-cadherin by HOXD3-overexpression are responsible for the enhanced motility and dissociation.