ABSTRACTPlant temperature‐induced lipocalins (TILs) have been shown to be responsive to heat stress (HS), but the nature of this response was unknown. In this study, a reverse genetic approach was taken to elucidate the role of Arabidopsis TIL1 (At5g58070) in thermotolerance. A T‐DNA knock‐out line of TIL1 (til1‐1) showed severe defects in basal (BT) and acquired thermotolerance (AT), which could be complemented by introducing the wild‐type gene. However, over‐expression of TIL1 did not significantly enhance thermotolerance in transgenic plants. TIL1 is peripherally associated with plasma membrane. Transcriptomic analysis showed that the heat shock response in til1‐1 seedlings was about the same as in the wild‐type plants except the expression of TIL1. The level of TIL1 did not affect the temperature threshold for heat shock protein induction. Ion leakage analysis revealed no significant difference in membrane stability between the wild‐type and til1‐1 seedlings. These results suggest that TIL1 is not involved in regulating membrane fluidity or stability. Nevertheless, the mutant plants were also more sensitive than the wild type to tert‐butyl hydroperoxide, a reagent that induces lipid peroxidation. Taken together, these data indicate that TIL1 is an essential component for thermotolerance and probably functions by acting against lipid peroxidation induced by severe HS.