Manumycin A (MA) is a well-tolerated natural antibiotic showing pleiotropic anticancer effects in various preclinical in vitro and in vivo models. Anticancer drugs may themselves act as stressors to induce the cellular adaptive mechanism that can minimize their cytotoxicity. Heat shock proteins (HSPs) as cytoprotective factors can counteract the deleterious effects of various stressful stimuli. In this study, we examined whether the anticancer effects of MA can be counteracted by the mechanism related to HSPs belonging to the HSPA (HSP70) family. We found that MA caused cell type-specific alterations in the levels of HSPAs. These changes included concomitant upregulation of the stress-inducible (HSPA1 and HSPA6) and downregulation of the non-stress-inducible (HSPA2) paralogs. However, neither HSPA1 nor HSPA2 were necessary to provide protection against MA in lung cancer cells. Conversely, the simultaneous repression of several HSPA paralogs using pan-HSPA inhibitors (VER-155008 or JG-98) sensitized cancer cells to MA. We also observed that genetic ablation of the heat shock factor 1 (HSF1) transcription factor, a main transactivator of HSPAs expression, sensitized MCF7 cells to MA treatment. Our study reveals that inhibition of HSF1-mediated heat shock response (HSR) can improve the anticancer effect of MA. These observations suggest that targeting the HSR- or HSPA-mediated adaptive mechanisms may be a promising strategy for further preclinical developments.