In this study, we report the potential of cannabidiol, one of the major cannabis constituents, for enhancing osteoblastic differentiation in U2OS and MGâ63 cells. Cannabidiol increased the expression of Angiopoietin1 and the enzyme activity of alkaline phosphatase in U2OS and MGâ63. Invasion and migration assay results indicated that the cell mobility was activated by cannabidiol in U2OS and MGâ63. Western blotting analysis showed that the expression of tight junction related proteins such as Claudin1, Claudin4, Occuludin1, and ZO1 was increased by cannabidiol in U2OS and MGâ63. Alizarin Red S staining analysis showed that calcium deposition and mineralization was enhanced by cannabidiol in U2OS and MGâ63. Western blotting analysis indicated that the expression of osteoblast differentiation related proteins such as distalâless homeobox 5, bone sialoprotein, osteocalcin, type I collagen, Runtârelated transcription factor 2 (RUNX2), osterix (OSX), and alkaline phosphatase was time dependently upregulated by cannabidiol in U2OS and MGâ63. Mechanistically, cannabidiolâregulated osteoblastic differentiation in U2OS and MGâ63 by strengthen the proteinâprotein interaction among RUNX2, OSX, or the phosphorylated p38 mitogenâactivated protein kinase (MAPK). In conclusion, cannabidiol increased Angiopoietin1 expression and p38 MAPK activation for osteoblastic differentiation in U2OS and MGâ63 suggesting that cannabidiol might provide a novel therapeutic option for the bone regeneration.