The reciprocal fate decision of mesenchymal stem cells (MSCs) to either bone or adipocytes is determined by Wnt-related signaling and the glucagon-like peptide-1 receptor (GLP-1R). Azoramide, an ER stress alleviator, was reported to have an antidiabetic effect. In this study, we investigated the function of azoramide in regulating the lineage determination of MSCs for either adipogenic or osteogenic differentiation. Microcomputed tomography and histological analysis on bone morphogenetic protein (BMP)2-induced parietal periosteum bone formation assays, C3H10T1/2 and mouse bone marrow MSC-derived bone formation and adipogenesis assays, and specific staining for bone tissue and lipid droplets were used to evaluate the role of azoramide on the lineage determination of MSC differentiation. Cells were harvested for Western blot and quantitative real-time polymerase chain reaction (PCR), and immunofluorescence staining was used to explore the potential mechanism of azoramide for regulating MSC differentiation. Based on MSC-derived bone formation assays both in vivo and in vitro, azoramide treatment displayed a cell fate determining ability in favor of adipogenesis over osteogenesis. Further mechanistic characterizations disclosed that both the GLP-1R agonist peptide exendin-4 (Ex-4) and GLP-1R small interfering (si)RNA abrogated azoramide dual effects. Moreover, cAMP-protein kinase A (PKA)-mediated nuclear β-catenin activity was responsible for the negative function of azoramide on bone formation in favor of adipogenesis. These data provide the first evidence to show that azoramide may serve as an inhibitor against GLP-1R in MSC lineage determination.