The purpose of this study was to evaluate the mechanism of ROS-induced hyperthermic cell death in a colon cancer cell line. HT-29 colon cancer cells were exposed to heat (43 °C) in the presence of tert-butyl hydroperoxide (t-BOOH). t-BOOH combined with hyperthermia significantly decreased cell viability as compared with t-BOOH or hyperthermia alone. This decrease in cell numbers was associated with retardation in the S phase transit and not through apoptosis. Cell death was noted to be accompanied by specific features characteristic of autophagy: the presence of cytoplasmic autophagic vacuoles; autophagosome membrane association of microtubule-associated protein light chain 3; accumulation of acidic vesicular organelles; and increased incorporation of MDC in the autophagosome. Thermal sensitization through modulation of cellular ROS may represent a novel approach to increase the efficacy of hyperthermia as an anticancer modality.