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 is largely unclear. We found that cultured mouse microglial cells preferentially express the SOC channel subunit Orai1, whereas the isoforms Orai2 and Orai3 are less abundant. From experiments on wildtype and knockout STIM1 and STIM2 mice, we provide evidence that both STIM proteins contribute to microglial SOCE. STIM1^-/- microglia showed a strong reduction in SOCE, whereas in STIM2^-/- cells this effect was smaller. ATP- or UDP-induced Ca²⁺ entry was also nearly abolished in STIM1^-/- microglia and clearly reduced in the absence of STIM2. Patch-clamp experiments showed a suppression of SOC current in STIM1-deficient microglia, whereas an Orai-like current developed in wild-type cells upon pipette application of inositol 1,4,5-trisphosphate. ATP-induced migration was reduced in the absence of either STIM1 or STIM2 or in the presence of different SOCE blockers (LaCl₃, 2-APB and ACA). Our data demonstrate that STIM1 and STIM2 are essential modulators of SOCE and are important for nucleotide-induced Ca²⁺ signals as well as ATP-induced migration in microglia.