Adenoviral vectors based on adenovirus type 5 (Ad5) are commonly used for gene therapy. The Ad5 fiber-knob region primarily interacts with the coxsackievirus and adenovirus receptor (CAR). Reportedly, when stimulated, this receptor participates in the regulation of cell-to-cell adhesion and cell migration. In oncogene therapy, cell migration can have adverse effects by promoting metastasis and infiltration. Alternatively, cell migration may enhance the therapeutic effect of gene therapy by promoting the healing of injured tissues. However, the effect of binding of the Ad fiber-knob region to CAR of target cells has not been investigated in detail. Therefore, the aim of the present study was to investigate the effects of the Ad5 vectors on cell migration with the use of wound healing and migration assays. The results showed that infection with the Ad5 vectors promoted the migration of A549 cells, as determined quantifiably. Furthermore, when the Ad5 fiber-knob protein was applied to A549 cells, the same results were obtained. Together, the results revealed that binding of the Ad fiber-knob protein to CAR causes cell migration as a functional change in target cells. Studying the effect of the Ad fiber-knob protein will lead to the development of a gene transfer vector with greater safety and therapeutic effects.