Microglial cells are the resident macrophages that are involved in brain injuries and infections. Recent studies using transcranial two-photon microscopy have shown that ATP and P2Y receptors mediated rapid chemotactic responses of miroglia to local injury. However, the molecular mechanism for microglial chemotaxis toward ATP is still unknown. To address this question, we employed a combination of simultaneous perforated whole-cell recordings and time-lapse confocal imaging in GFP-labeled microglia in acute brain slices from adult mice. We found that ATP-induced rapid chemotaxis is correlated with P2Y receptor associated-outward potassium current in microglia. Activation of both P2Y receptor and its associated potassium channels are required for ATP-induced chemotaxis and baseline motility of microglial cells. The chemotaxis required the activation of phosphoinositide 3-kinase but not mitogen-activated protein kinase pathway. Our results provide strong evidence that P2Y receptor-associated outward potassium channels and the phosphoinositide 3-kinase pathway are important for ATP-induced microglial motility in acute brain slices.