We have previously demonstrated that interrupting the protein-protein interaction (PPI) of β-tubulin:chaperonin-containing TCP-1β (CCT-β) induces the selective killing of multidrug-resistant cancer cells due to CCT-β overexpression. However, the molecular mechanism has not yet been identified. In this study, we found that CCT-β interacts with a myriad of intracellular proteins involved in the cellular functions of the endoplasmic reticulum (ER), mitochondria, cytoskeleton, proteasome and apoptosome. Our data show that the targeted cells activate both the heat-shock protein 90 (Hsp90)-associated protein ubiquitination/degradation pathway to eliminate misfolded proteins in the cytoplasm and the valosin-containing protein (VCP)-centered ER-associated protein degradation pathway to reduce the excessive levels of unfolded polypeptides from the ER, thereby mitigating ER stress, at the onset of β-tubulin:CCT-β complex disruption. Once ER stress is expanded, ER stress-associated apoptotic signaling is enforced, as exhibited by cellular vacuolization and intracellular Ca2+ release. Furthermore, the elevated intracellular Ca2+ levels resulting from capacitative Ca2+ entry augments apoptotic signaling by provoking mitochondrial perturbation and caspase overactivation in the targeted cells. These findings not only provide a detailed picture of the apoptotic signaling cascades evoked by targeting the β-tubulin:CCT-β complex but also demonstrate a strategy to combat malignancies with chemoresistance to Hsp90- and VCP-related anticancer agents.