Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase implicated in cell cycle progression and cell migration. Overexpression of FAK in a variety of tumors has suggested that FAK is a promising target for therapeutic intervention. In this study, we took advantage of a modified polyethylenimine (M-PEI) with high transfection efficiency for tumor cells and tissues, and targeted FAK function through both in vitro and in vivo approaches. The results demonstrated that both plasmid-encoded FAK small interfering RNA (siRNA) and overexpression of FAK-related non-kinase (FRNK, FAK dominant negative) dramatically inhibited in vitro B16F10 cell proliferation and invasion. We used two transplantable mouse tumor models of primary and metastatic melanoma to evaluate the therapeutic potential of PEI-complexed plasmids targeting FAK function. The results revealed that intratumoral delivery of PEI-complexed plasmids targeting FAK significantly suppressed primary tumor growth as well as metastasis of B16F10 cells into lung and lymph nodes. Both approaches prolonged the survival of the tumor-bearing mice. Taken together, these results indicate that intratumoral delivery of plasmid DNA targeting FAK function, using M-PEI as a gene carrier, represents a promising avenue for melanoma therapy.