We previously reported that extracellular ATP induces membrane ruffling and chemotaxis of microglia and suggested that their induction is mediated by the Gi/o‐protein coupled P2Y₁₂ receptor (P2Y₁₂R). Here we report discovering that the P2X₄ receptor (P2X₄R) is also involved in ATP‐induced microglial chemotaxis. To understand the intracellular signaling pathway downstream of P2Y₁₂R that underlies microglial chemotaxis, we examined the effect of two phosphatidylinositol 3′‐kinase (PI3K) inhibitors, wortmannin, and LY294002, on chemotaxis in a Dunn chemotaxis chamber. The PI3K inhibitors significantly suppressed chemotaxis without affecting ATP‐induced membrane ruffling. ATP stimulation increased Akt phosphorylation in the microglia, and the increase was reduced by the PI3K inhibitors and a P2Y₁₂R antagonist. These results indicate that P2Y₁₂R‐mediated activation of the PI3K pathway is required for microglial chemotaxis in response to ATP. We also found that the Akt phosphorylation was reduced when extracellular calcium was chelated, suggesting that ionotropic P2X receptors are involved in microglial chemotaxis by affecting the PI3K pathway. We therefore tested the effect of various P2X₄R antagonists on the chemotaxis, and the results showed that pharmacological blockade of P2X₄R significantly inhibited it. Knockdown of the P2X₄ receptor in microglia by RNA interference through the lentivirus vector system also suppressed the microglial chemotaxis. These results indicate that P2X₄R as well as P2Y₁₂R is involved in ATP‐induced microglial chemotaxis. © 2007 Wiley‐Liss, Inc.