The functional expression of the G protein–coupled P2Y₂ nucleotide receptor (P2Y₂R) has been associated with proliferation and migration of vascular smooth muscle cells (SMCs), 2 processes involved in atherosclerosis and restenosis. Activation of the P2Y₂R causes dynamic reorganization of the actin cytoskeleton, which transmits biochemical signals and forces necessary for cell locomotion, suggesting that P2Y₂Rs may be linked to the actin cytoskeleton. Here, we identified filamin A (FLNa) as a P2Y₂R-interacting protein using a yeast 2-hybrid system screen with the C-terminal region of the P2Y₂R as bait. The FLNa binding site in the P2Y₂R is localized between amino acids 322 and 333. Deletion of this region led to selective loss of FLNa binding to the P2Y₂R and abolished Tyr phosphorylation of FLNa induced by the P2Y₂R agonist UTP. Using both time-lapse microscopy and the Transwell cell migration assay, we showed that UTP significantly increased SMC spreading on collagen I (6.8 fold; P0.01) and migration (3.6 fold; P0.01) of aortic SMCs isolated from wild-type mice, as compared with unstimulated SMCs. UTP-induced spreading and migration of aortic SMCs did not occur with cells isolated from P2Y₂R knockout mice. Expression of the full-length P2Y₂R in SMCs isolated from P2Y₂R knockout mice restored both UTP-induced spreading and migration. In contrast, UTP-induced spreading and migration did not occur in SMCs isolated from P2Y₂R knockout mice transfected with a mutant P2Y₂R that does not bind FLNa. Furthermore, ex vivo studies showed that both ATP and UTP (10 µmol/L) promoted migration of SMCs out of aortic explants isolated from wild-type but not P2Y₂R knockout mice. Thus, this study demonstrates that P2Y₂R/FLNa interaction selectively regulates spreading and migration of vascular SMCs.