<p>Over the past few decades, microtubules have been targeted by various anticancer drugs, including paclitaxel and eribulin. Despite their promising effects, the development of drug resistance remains a challenge. We aimed to define a novel cell death mechanism that targets microtubules using eribulin and to assess its potential in overcoming eribulin resistance. Notably, treating non-resistant breast cancer cells with eribulin led to increased microtubule acetylation around the nucleus and cell death. Conversely, eribulin-resistant (EriR) cells did not exhibit a similar increase in acetylation, even at half-maximal inhibitory concentrations. Interestingly, silencing the <i>ATAT1</i> gene, which encodes the α-tubulin N-acetyltransferase 1 (the enzyme responsible for microtubule acetylation), induces eribulin resistance, mirroring the phenotype of EriR cells. Moreover, eribulin-induced acetylation of microtubules facilitates the transport of Ca²⁺ from the ER to the mitochondria, releasing cytochrome c and subsequent cell death. Transcriptome analysis of EriR cells revealed a significant downregulation of ER stress-induced apoptotic signals, particularly the activity of protein kinase RNA-like ER kinase (PERK), within the unfolded protein response signaling system. Pharmacological induction of microtubule acetylation through a histone deacetylase 6 inhibitor combined with the activation of PERK signaling using the PERK activator CCT020312 in EriR cells enhanced mitochondrial Ca²⁺ accumulation and subsequent cell death. These findings reveal a novel mechanism by which eribulin-induced microtubule acetylation and increased PERK activity lead to Ca²⁺ overload from the ER to the mitochondria, ultimately triggering cell death. This study offers new insights into strategies for overcoming resistance to microtubule-targeting agents.</p>