Purpose: The purpose of the present study was to evaluate the efficacy of mild hyperthermia to potentiate the anticancer effects of β-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-6]pyran-5,6-dione) by up-regulating NAD(P)H:quinone oxidoreductase (NQO1) in cancer cells. Experimental Design: Effects of β-apachone alone or in combination with mild heating on the clonogenic survival of FSall fibrosarcoma cells of C3H mice and A549 human lung tumor cells in vitro was determined. Effects of heating on the NQO1 level in the cancer cells in vitro were assessed using Western blot analysis for NQO1 expression, biochemical determination of NQO1 activity, and immunofluorescence microscopy for NQO1 expression. Growth of FSall tumors in the hind legs of C3H mice was determined after treating the host mice with i.p. injection of 45 mg/kg β-lapachone followed by heating the tumors at 42°C for 1 hour every other day for four times. Results: Incubation of FSall tumor cells and A549 tumor cells with β-lapachone at 37°C reduced clonogenic survival of the cells in dose-dependent and incubation time - dependent manner. NQO1 level in the cancer cells in vitro increased within 1 hour after heating at 42°C for 1 hour and remained elevated for >72 hours. The clonogenic cell death caused by β-lapachone increased in parallel with the increase in NQO1 levels in heated cells. Heating FSall tumors in the legs of C3H mice enhanced the effect of i.p.-injected β-lapachone in suppressing tumor growth. Conclusion: We observed for the first time that mild heat shock up-regulates NQO1 in tumor cells. The heat-induced up-regutation of NQO1 enhanced the anticancer effects of β-lapachone in vitro and in vivo.