Store-operated Ca2+ entry (SOCE) is established by formation of subplasmalemmal clusters of the endoplasmic reticulum (ER) protein, stromal interacting molecule 1 (STIM1) upon ER Ca2+ depletion. Thereby, STIM1 couples to plasma membrane channels such as Orai1. Thus, a close proximity of ER domains to the plasma membrane is a prerequisite for SOCE activation, challenging the concept of local Ca2+ buffering by mitochondria as being essential for SOCE. This study assesses the impact of mitochondrial Ca2+ handling and motility on STIM1–Orai1-dependent SOCE. High-resolution microscopy showed only 10% of subplasmalemmal STIM1 clusters to be colocalized with mitochondria. Impairments of mitochondrial Ca2+ handling by inhibition of mitochondrial Na+-Ca2+ exchanger (NCXmito) or depolarization only partially suppressed Ca2+ entry in cells overexpressing STIM1-Orai1. However, SOCE was completely abolished when both NCXmito was inhibited and the inner mitochondrial membrane was depolarized, in STIM1- and Orai1-overexpressing cells. Immobilization of mitochondria by expression of mAKAP-RFP-CAAX, a construct that physically links mitochondria to the plasma membrane, affected the Ca2+ handling of the organelles but not the activity of SOCE. Our observations indicate that mitochondrial Ca2+ uptake, including reversal of NCXmito, is fundamental for STIM1–Orai1-dependent SOCE, whereas the proximity of mitochondria to STIM1-Orai1 SOCE units and their motility is not required.