Heat‐induced cell death appears to be a cell‐specific event. Chronic heat stress was lethal to human colon cancer cells (Caco‐2, HT29, and HCT116), but not to normal diploid fibroblasts and other cancer cells (BJ‐T, WI38, HeLa, ovarian 2008, WI38VA). Acute heat stress (45–51°C, 30 min) caused cell death of colon cancer cells during recovery at physiological temperature. Thermal killing of Caco‐2 cells was not mediated via oxidative stress since Caco‐2 cells were much more resistant than HeLa and other cancer cells to H₂O₂‐induced cell death. Acute heat stress caused a striking loss of eukaryotic initiation factor 5A (eIF5A) in colon cancer cells, but not in HeLa and other normal or transformed human fibroblasts. The heat‐induced loss of eIF5A is likely to be due to changes in the protein stability. The half‐life of eIF5A was changed from >20 h to less than 30 min during the acute heat stress. Sequence analysis of the eIF5A gene from Caco‐2 and HeLa cells did not reveal any difference, suggesting that the change in stability in Caco‐2 cells was not due to any eIF5A mutation. Pretreatment of cells with protease inhibitors such as phenylmethyl sulfonyl fluoride (PMSF) partially blocked the heat‐induced loss of eIF5A and prevented heat‐induced cell death. In light of the essential role of eIF5A in cell survival and proliferation, our results suggest that the stability of eIF5A may have an important role in determining the fate of the particular cell type after severe heat stress. J. Cell. Physiol. 219: 485–493, 2009. © 2009 Wiley‐Liss, Inc.