Adaptation to endoplasmic reticulum (ER) stress through the upregulation of the ER chaperone BiP/Grp78 favors resistance of cancer cells to anthracyclins. We recently demonstrated that the mitochondrial HSP90 chaperone TNF receptor-associated protein 1 (TRAP1) is also localized in the ER, where it is responsible for protection from ER stress and quality control on specific mitochondrial proteins contributing to its anti-apoptotic function and the regulation of the mitochondrial apoptotic pathway. Based on the evidence that Bip/Grp78 and TRAP1 are co-upregulated in about 50% of human breast carcinomas (BCs), and considering that the expression of TRAP1 is critical in favoring resistant phenotypes to different antitumor agents, we hypothesized that ER-associated TRAP1 is also favoring resistance to anthracyclins. Indeed, anthracyclins induce ER stress in BC cells and cross-resistance between ER stress agents and anthracyclins was observed in bortezomib- and anthracyclin-resistant cells. Several lines of evidence suggest a mechanistic link between the ER-stress protecting function of TRAP1 and resistance to anthracyclins: i) ER stress- and anthracyclin-resistant cell lines are characterized by the upregulation of TRAP1; ii) TRAP1 silencing in both drug-resistant cell models restored the sensitivity to bortezomib and anthracyclins; iii) the transfection of a TRAP1 deletion mutant, whose localization is restricted to the ER, in TRAP1 KD cells protected from apoptosis induced by anthracyclins; iv) the disruption of the ER-associated TRAP1/TBP7 pathway by a TBP7 dominant negative deletion mutant re-established drug sensitivity in drug-resistant cells. This process is likely mediated by the ability of TRAP1 to modulate the PERK pathway as TRAP1 KD cells failed to induce the phosphorylation of PERK in response to anthracyclins. Moreover, the downregulation of TRAP1 in combination with ER stress agents produced high cytotoxic effects in BC cells. These results suggest that ER-associated TRAP1 plays a role in protecting tumor cells against DNA damaging agents by modulating the PERK pathway.