With the increase of environment temperature, more and more attentions are payed to the effects of heat stress. Cells under heat shock either are adapted to the condition or are damaged and dead. In this paper, we found that heat shock induced endoplasmic reticulum (ER) stress. ATF4, PERK, and IRE1α were induced by heat shock of 45 °C in the transcriptional level. Under the stress of 45 °C, PERK was phosphorylated and XBP1s was detected. The result indicated that heat shock could induce the ER stress. We found that heat shock of 45 °C induced the dysregulation of HSP70 and DNA-PKcs, and downregulated the expression of PARP1 and XRCC1. Further results showed that after the knockdown of ATF4 or IRE1α, the expression of DNA-PKcs and XRCC1 were increased. It was indicated that ATF4 and IRE1α could inhibit the expression of DNA-PKcs and XRCC1 under the heat stress. Our results suggested that heat shock could activate ER stress. IRE1α and ATF4, as the important ER stress molecules, could inhibit the expression of DNA repair proteins DNA-PKcs, XRCC1, and HSP70 under heat shock. Downregulation of DNA repair proteins could aggravate the cell damage that may cause cell apoptosis. This may explain that heat shock could increase the lethality of chemotherapeutic drugs on tumor cells.