Single exposure of cells to hyperthermic treatment, makes them thermotolerant to subsequent heat treatment. Tumor cells that express normal or elevated levels of HSPs show thermotolerence. KNK437, a chemically synthesized benzylidene lactam compound, has been specifically found to inhibit the synthesis of heat shock proteins and thus leading to significant reduction in the thermotolerance of cells. However, mechanisms by which KNK437 inhibits thermotolerence are still not clearly understood. In this study an attempt has been made to elucidate the mechanisms of inhibition of HSPs by KNK437 in 2 cell lines of human oral squamous cell carcinma with varying sensitivities (HSC4 ; heat-resistant and KB ; heat-sensitive) with an aim to identify which of the HSPs are targeted by KNK437. Single administration of 100μM KNK437-treatment always resulted in a higher than 85% cell survival compared to the untreated groups, indicating the KNK437 had a minimum cytotoxic effect in both cell lines. Combination of hyperthermia with KNK437 after heat shock, specifically inhibited the induction of HSPs, where the expression of HSP27, HSP40, HSP70 and HSP90 reduced in a time dependent manner. Interestingly, the hyperthermic treatment combined with KNK437 produced supra-reductive effect on the expression of HSP27 and HSP90 in HSC4, HSP27, HSP40 and HSP90 in KB cells, when compared to heat treatment alone. Our results indicate that inhibition of HSPs by KNK437 plays an important role in inhibiting the thermotolerence of the cells and may be a valuable to improve the efficacy of oral cancer during hyperthermic therapy.