Background: Microtubule polymerization is usually considered as the upstream of apoptotic cell death induced by taxanes, but recently published studies provide more insights into the mechanisms responsible for the antineoplastic effect of taxanes. In this study, we figure out the role of the stress-related PERK/eIF2α axis in tumor cell death upon taxane treatment along with paclitaxel resistance. Methods: Utilizing immunoblot assay, the activation status of PERK-eIF2α signaling was detected in a panel of cancer cell lines after the treatment of taxanes. The causal role of PERK-eIF2α signaling in the cancer cell apoptosis induced by taxanes was examined via pharmacological and genetic inhibitions of PERK. The relationship between microtubule polymerization and PERK-eIF2α activation was explored by immunofluorescent and immunoblotting assays. Eventaually, the combined therapeutic effect of paclitaxel (PTX) and CCT020312, a PERK agonist, was investigated in PTX-resistant breast cancer cells in vitro and in vivo. Results: PERK-eIF2α axis was dramatically activated by taxanes in several cancer cell types. Pharmacological or genetic inhibition of PERK efficiently impaired taxane-induced apoptotic cell death, independent of the cellular microtubule polymerization status. Moreover, PTX was able to activate the PERK/eIF2α axis in a very low concentration without triggering microtubule polymerization. In PTX-resistant breast cancer cells, the PERK/eIF2α axis was attenuated in comparison with the PTX-sensitive counterparts. Reactivation of the PERK/eIF2α axis in the PTX-resistant breast cancer cells with PERK agonist sensitized them to PTX in vitro. Combination treatment of the xenografted PTX-resistant breast tumors with PERK agonist and PTX validated the synergic effect of PTX and PERK activation in vivo. Conclusion: Activation of the PERK/eIF2α axis is a pivotal prerequisite of taxanes to initiate cancer cell apoptosis, which is independent of the well-known microtubule polymerization-dependent manner. Simultaneous activation of PERK-eIF2α signaling would be a promising therapeutic strategy to overcome PTX resistance in breast cancer or other cancers.