Osteoporosis (OP) is a systemic skeletal disease leading to fragility fractures and is a major health issue globally. WNT/β-catenin signaling regulates bone-remodeling processes and plays vital roles in OP development. However, the underlying regulatory mechanisms behind WNT/β-catenin signaling in OP requires clarification, as further studies are required to identify novel alternate therapeutic agents to improve OP. Here we report that fibronectin 1 (FN-1) promoted differentiation and mineralization of osteoblasts by activating WNT/β-catenin pathway, in cultured pre-osteoblasts. With isobaric tags for relative and absolute quantitation labeling proteomics analysis, we investigated protein changes in bone samples from OP patients and normal controls. FN-1 accumulated in osteoblasts in bone samples from OP patients and age-related OP mice compared to control group. In addition, we observed that integrin β1 (ITGB1) acts as an indispensable signaling molecule for the interplay between FN-1 and β-catenin, and that FN-1 expression increased, but ITGB1 expression decreased in osteoblasts during OP progression. Therefore, our study reveals a novel explanation for WNT/β-catenin pathway inactivation in OP pathology. Supplying of FN-1 and ITGB1 may provide a potential therapeutic strategy in improving bone formation during OP.