Heat shock factor 1 (HSF1) is a transcription factor essential for tumorigenesis, and targeting HSF1 may be effective in combined therapeutics for cervical cancer. Cyclosporin A (CsA) is an immunosuppressant that has revolutionized organ transplantation. However, the roles and regulatory mechanisms by which CsA modulates HSP expression remain largely unknown. In this study, we found that CsA pretreatment prevented induction of HSPs during heat shock by enhancing the phosphorylation of Ser303 and Ser307 on HSF1 and thus inhibiting its transcriptional activity. Suppression of ERK1/2, GSK3β and CK2 activities attenuated CsA-induced down-regulation of HSP expression and up-regulation of HSF1 phosphorylation. CsA interfered with HSF1-SSBP1 complex formation and HSF1 nuclear translocation and recruitment to the HSP70 promoter. CsA clearly caused HeLa cell death during proteotoxic stress through reduced expression of HSPs. These results indicate that CsA suppresses HSP induction during heat shock by regulating the phosphorylation and nuclear translocation of HSF1. Our results provide a conceptual framework for the development of novel therapeutic strategies for cervical cancer through application of CsA during hyperthermia or chemotherapy.