The endoplasmic reticulum-resident Ca²⁺ sensors STIM1 and STIM2 trigger store-operated Ca²⁺ (SOC) entry by activation of SOC channels in the plasma membrane. However, the differential role of STIM sensors and the identity of SOC channels in microglia are largely unclear. In qPCR experiments we found a preferential expression of the SOC channel subunit ORAI1, whereas the isoforms ORAI2 and ORAI3 are less abundant. From experiments on wildtype and knockout STIM1, STIM2 and Orai1 mice, we provide evidence that both STIM isoforms contribute to SOC entry and that Orai1 is the main SOC channel subunit in cultured mouse microglia. Orai1^-/- and STIM1^-/- microglia showed a strong reduction in SOC entry, whereas in STIM2^-/- cells this effect was smaller. Ca²⁺ entry evoked by the extracellular nucleotides 2-MeSADP and UDP were nearly complete suppressed in STIM1^-/- and in Orai^-/- microglia and reduced in cells lacking STIM2. Chemotactic responses to the specific P2Y₁₂ receptor agonist 2-MeSADP were inhibited in the absence of STIM1 or STIM2. Phagocytosis and migration induced by the P2Y₆ receptor agonist UDP were reduced in both STIM1^-/- and STIM2^-/- cells. Our data demonstrate that both ER Ca²⁺ sensors are essential for SOC entry, purinergic Ca²⁺ signals as well nucleotide-induced migration and phagocytosis in microglia.