ARF6 GTPase is a conserved regulator of membrane trafficking and actin-based cytoskeleton dynamics at the leading edge of migrating cells. A key determinant of ARF6 function is the lifetime of the GTP-bound active state, which is orchestrated by GTPase-activating protein (GAP) and GTP-GDP exchanging factor. However, very little is known about the molecular mechanisms underlying ARF6-mediated cell migration. To systematically analyze proteins that regulate ARF6 activity during cell migration, we perform a proteomic analysis of proteins selectively bound to active ARF6 using mass spectrometry and identify a novel ARF6-specific GAP, ACAP4. ACAP4 encodes 903 amino acids and contains two coiled coils, one PH domain, one GAP motif, and two ankyrin repeats. Our biochemical characterization demonstrates that ACAP4 has a PIP2-dependent GAP activity specific for ARF6. The co-localization of ACAP4 with ARF6 occurs in ruffling membranes formed upon AIF4 and EGF stimulation. ACAP4 overexpression limits the recruitment of ARF6 to the membrane ruffles in the absence of EGF stimulation. Expression of GTP-hydrolysis resistant ARF6Q67L results in accumulations of ACAP4 and ARF6 in the cytoplasmic membrane, suggesting that GTP hydrolysis is required for the ARF6-dependent membrane remodeling. Significantly, the depletion of ACAP4 by siRNA or inhibition of ARF6 GTP hydrolysis by overexpressing GAP-deficient ACAP4 suppresses ARF6-dependent cell migration in wound-healing, demonstrating the importance of ACAP4 in cell migration. Thus, our study sheds new light on the biological function of ARF6-mediated cell migration.