Stress-induced cellular defense machinery has a critical role in mediating cancer drug resistance, and targeting stress-related signaling has become a novel strategy to improve chemosensitivity. Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid with potent anticancer bioactivities in multiple malignancies, whereas its underlying mechanisms remain unclear. Here in, we found that BA has synergistic effects with taxol to induce breast cancer cells G2/M checkpoint arrest and apoptosis induction, but had little cytotoxicity effects on normal mammary epithelial cells. Drug affinity responsive target stability (DARTS) strategy further identified glucose-regulated protein 78 (GRP78) as the direct interacting target of BA. BA administration significantly elevated GRP78-mediated endoplasmic reticulum (ER) stress and resulted in the activation of protein kinase R-like ER kinase (PERK)/eukaryotic initiation factor 2a/CCAAT/enhancer-binding protein homologous protein apoptotic pathway. GRP78 silencing or ER stress inhibitor salubrinal administration was revealed to abolish the anticancer effects of BA, indicating the critical role of GRP78 in mediating the bioactivity of BA. Molecular docking and coimmunoprecipitation assay further demonstrated that BA might competitively bind with ATPase domain of GRP78 to interrupt its interaction with ER stress sensor PERK, thereby initiating the downstream apoptosis cascade. In vivo breast cancer xenografts finally validated the chemosensitizing effects of BA and its biofunction in activating GRP78 to trigger ER stress-mediated apoptosis. Taken together, our study not only uncovers GRP78 as a novel target underlying the chemosensitizing effects of BA, but also highlights GRP78-based targeting strategy as a promising approach to improve breast cancer prognosis.