Hyperthermia, as an adjuvant therapy, has shown promising anti‐tumor effects. Ovarian tumor domain‐containing 7B (OTUD7B) is a deubiquitinating enzyme that is frequently found in a variety of cancers. The aim of this study is to investigate the role of OTUD7B in lung cancer hyperthermia and the underlying mechanism. A549 and CALU‐3 cells were respectively exposed to 42 or 44°C for the indicated times (0, 1, 3, or 6 h) followed by incubation at 37°C for 24 h. We found a temperature‐ and time‐dependent decrease in cell viability and an increase in apoptosis levels. Compared with 0 h, heat treatment for 3 h inhibited the proliferation and invasion of A549 cells, reduced the expression levels of mitochondrial membrane potential, IAP family members (cIAP‐1 and XIAP) proteins and ubiquitination of Smac, and increased Smac protein expression. Treatment with 10 μM Smac mimic BV6 further enhanced the anti‐tumor effect of hyperthermia. Next, co‐IP validation showed that OTUD7B interacted with Smac and stabilized Smac through deubiquitination. OTUD7B overexpression induced damage in A549 and CALU‐3 cells, while silencing OTUD7B caused opposite effects. Overexpressing OTUD7B enhanced the anti‐cancer effect of hyperthermia, while si‐OTUD7B reversed the anti‐cancer effect of hyperthermia, which was verified in the xenograft tumor model in nude mice. Taken together, OTUD7B may serve as a potential anticancer factor with potential clinical efficacy in the thermotherapeutic treatment of lung cancer.
