Introduction: Today, cancer stem cells have attracted a lot of attention as tumour-initiating cells. Hyperthermia was also found to be beneficial in cancer treatment in many studies. In the present study, we assessed the effects of hyperthermia on colorectal cancer cells (HT-29) and their derived cancer stem cells (CSCs). Materials and Methods: CSCs were isolated from the HT-29 cell lines and then, the expression of surface marker and stemness-related genes were evaluated. Next, the toxicity effect of hyperthermia on HT-29 and CSCs was assessed. In addition, the effect of hyperthermia on the expression of stemness, proliferation, Wnt signaling pathway, and apoptosis genes was evaluated. Finally, the data were statistically analyzed. Results: Isolated-CSCs were positive for CD133 surface marker and the expression of stemness genes in the isolated-CSCs was significantly higher than HT-29 cells (P-value< 0. 05). Treatment with hyperthermia reduced the survival rate of HT-29 and CSCs. In both groups, the gene expression of OCT3/4, NANOG, PCNA, WNT1, and CTNNB1, was decreased (P>0. 05). In addition, the expression of P53, KLF4, and BAX genes was significantly increased in CSCs (P <0. 05). Conclusion: The results showed that hyperthermia did not have a significant effect on HT-29 cells. However, it had a significant effect on the cell viability, growth curve, expression of genes involved in apoptosis and KLF4 in treated-CSCs. Therefore, hyperthermia can have a beneficial effect on inhibiting the growth and proliferation of CSCs.