Hyperthermia has been used as an adjuvant treatment for radio- and chemotherapy for decades. In addition to its effects on perfusion and oxygenation of cancer tissues, hyperthermia can enhance the efficacy of DNA-damaging treatments such as radiotherapy and chemotherapy. Although it is believed that the adjuvant effects are based on hyperthermia-induced dysfunction of DNA repair systems, the mechanisms of these dysfunctions remain elusive. Here, we propose that elevated temperatures can induce chromatin trapping (-trapping) of essential factors, particularly those involved in DNA repair, and thus enhance the sensitization of cancer cells to DNA-damaging therapeutics. Using mass spectrometry-based proteomics, we identified proteins that could potentially undergo -trapping in response to hyperthermia. Functional analyses of several identified factors involved in DNA repair demonstrated that -trapping could indeed be a mechanism of hyperthermia-induced transient deficiency of DNA repair systems. Based on our proteomics data, we showed for the first time that hyperthermia could inhibit maturation of Okazaki fragments and activate a corresponding poly(ADP-ribose) polymerase-dependent DNA damage response. Together, our data suggest that chromatin trapping of factors involved in DNA repair and replication contributes to heat-induced radio- and chemosensitization.