Protein synthesis inhibitors can suppress the development of thermotolerance in tumor tissues on repeated heating. Withaferin A (WA), isolated from Withania somnifera has cytotoxic and inhibitory action on protein synthesis. In the present investigation, effect of WA on development and decay of thermotolerance in B16F1 melanoma was studied in C57BL mice. Tumors of 10010 mm3 size were subjected to repeated hyperthermia (HT) at 43°C for 30 minutes. WA was injected after first hyperthermia treatment. The tumor response was assessed by calculating the tumor growth delay (GD). The GD increased with increase in time gap between two hyperthermia treatments and was significantly higher (p <0.05 to p <0.001) in WA treated groups at all the respective time gaps (except at 0h and 120h) compared to hyperthermia alone group. WA increases the tumor response during repeated hyperthermia by reducing the magnitude of thermotolerance developed and by decreasing the recovery time from thermotolerance. © 2009 SAGE Publications.