Activation of microglia is the first and main immune response to brain injury. Release of the nucleotides ATP, ADP, and UDP from damaged cells regulate microglial migration and phagocytosis via purinergic P2Y receptors. We hypothesized that store‐operated Ca2+ entry (SOCE), the prevalent Ca2+ influx mechanism in non‐excitable cells, is a potent mediator of microglial responses to extracellular nucleotides. Expression analyses of STIM Ca2+ sensors and Orai Ca2+ channel subunits, that comprise the molecular machinery of SOCE, showed relevant levels of STIM1, STIM2, and Orai1 in cultured mouse microglia. STIM1 expression and SOCE were down‐regulated by treatment of microglia with lipopolysaccharide, suggesting that inflammation limits SOCE by lower STIM1 abundance. Ca2+ entry induced by cyclopiazonic acid, ATP, the P2Y6 receptor agonist UDP, or the P2Y12 receptor agonist 2‐methylthio‐ADP (2‐MeSADP) was clearly affected in microglia from Stim1–/–, Stim2–/–, and Orai1–/– mice. SOCE blockers or ablation of STIM1, STIM2, or Orai1 severely impaired nucleotide‐induced migration and phagocytosis in microglia. Thus, this study assigns SOCE, regulated by STIM1, STIM2, and Orai1 an essential role in purinergic signaling and activation of microglia. GLIA 2015;63:652–663