The acquisition of MDR1-mediated chemoresistance poses a major obstacle to the success of conventional chemotherapeutic agents. HSF1 is also involved in chemoresistance, and several studies have demonstrated the relationship between HSF1 and MDR1 but without any consistent results. Paclitaxel- and doxorubicin-resistant cancer cells showed higher expression of MDR1 and HSF1. Depletion of HSF1 decreased mdr1 expression at mRNA level, and HSF1 directly interacted with the promoter site of mdr1, suggesting its role as a transcriptional regulator of MDR1. Phosphorylation of Ser303/307, which was involved in protein stability of HSF1 by FBXW7-mediated degradation, was found to be important for transcriptional activation of mdr1. Drug-resistant cells showed decreased expression of FBXW7, which was mediated by the activation of ERK1/2, thus indicating that over-activation of ERK1/2 in drug-resistant cells decreased FBXW7 protein stability, which finally inhibited protein degradation of pHSF1 at Ser303/307. There was a positive correlation between immunofluorescence data of pHSF1 at Ser303/307 and MDR1 in carcinogen-induced rat mammary tumors and human lung cancers. These findings identified the post-translational mechanisms of HSF1 transcription in MDR1 regulation of drug resistance development.