Microglia are immune effector cells in the central nervous system (CNS) and their activation, migration and proliferation play crucial roles in brain injuries and diseases. We examined the role of intracellular Ca2+-independent phospholipase A2 (iPLA2) in the regulation of microglia chemotaxis toward ADP. Inhibition of iPLA2 by 4-bromoenol lactone (BEL) or iPLA2 knockdown exerted a significant inhibition on phosphatidylinositol-3-kinase (PI3K) activation and chemotaxis. Further examination revealed that iPLA2 knockdown abrogated Src activation, which is required for PI3K activation and chemotaxis. Colocalization studies showed that cSrc-GFP was retained in the endosomal recycling compartment (ERC) in iPLA2 knockdown cells, but the addition of arachidonic acid (AA) could restore cSrc trafficking to the plasma membrane by allowing the formation/release of recycling endosomes associated with cSrc-GFP. Using BODIPY-AA, we showed that AA is selectively enriched in recycling endosomes. These results suggest that AA is required for the cSrc trafficking to the plasma membrane by controlling the formation/release of recycling endosomes from the ERC. © 2011 John Wiley & Sons A/S.