The SLIT-ROBO GTPase-activating proteins (srGAPs) are critical for neuronal migration through inactivation of Rho GTPases Cdc42, Rac1 and RhoA. Here we report that srGAP2 physically interacts with protein arginine methyltransferase 5 (PRMT5). srGAP2 localizes to the cytoplasm and plasma membrane protrusion. srGAP2 knockdown reduces cell spreading and increases cell migration, but has no effects on cell proliferation. PRMT5 binds to the N-terminal of srGAP2 (225-538 aa) and methylates its C-terminal arginine residue R927. The methylation mutant srGAP2-R927A fails to rescue cell spreading defect, is unable to localize to plasma membrane leading edge, and perturbs the srGAP2 homo-dimer formation mediated by the F-BAR domain. These results suggest that srGAP2 arginine methylation plays important roles on cell spreading and cell migration through influencing membrane protrusion.