Osteosarcoma is the most frequent and intractable malignancy of the bone in children and young adults. Surgical operation requires extensive excision of the cancer tissue and neighboring normal tissues. In addition, anticancer drugs and radiation therapy are thought to be almost ineffective. Glucose-regulated protein 78 (GRP78), a cell-protective endoplasmic reticulum (ER) chaperone protein, is one of the most promising anticancer targets for osteosarcoma. Here, by analyzing the molecular mechanisms of kuanoniamine C, we report that kuanoniamine C suppresses GRP78 expression via GRP78 mRNA degradation in an ER stress response-independent manner. Interestingly, kuanoniamine C-induced cell death and downregulation of GRP78 expression was regulated by p53 signaling. Moreover, co-treatment with bortezomib, which is a newly identified anticancer drug for osteosarcoma, and kuanoniamine C suppressed GRP78 protein expression, which is essential for the stimulation of bortezomib-induced cell death. These results suggest that co-treatment with bortezomib and kuanoniamine C is a novel therapeutic strategy for the treatment of osteosarcoma that enhances bortezomib-dependent cell death by the downregulation of GRP78, and this combination selectively targets the major cell population of osteosarcoma, which expresses wild-type p53.