Intracellular Ca²⁺ is essential for diverse cellular functions. Ca²⁺ entry into many cell types including immune cells is triggered by depleting endoplasmic reticulum (ER) Ca²⁺, a process termed store-operated Ca²⁺ entry (SOCE). STIM1 is an ER Ca²⁺ sensor. Upon Ca²⁺ store depletion, STIM1 clusters at ER–plasma membrane junctions where it interacts with and gates Ca²⁺-permeable Orai1 ion channels. Here we show that STIM1 is also activated by temperature. Heating cells caused clustering of STIM1 at temperatures above 35 °C without depleting Ca²⁺ stores and led to Orai1-mediated Ca²⁺ influx as a heat off-response (response after cooling). Notably, the functional coupling of STIM1 and Orai1 is prevented at high temperatures, potentially explaining the heat off-response. Additionally, physiologically relevant temperature shifts modulate STIM1-dependent gene expression in Jurkat T cells. Therefore, temperature is an important regulator of STIM1 function.