α2-antiplasmin (α2AP) is known to be a physiological inhibitor of plasmin. Previously, we showed that α2AP displays various functions, such as promotion of extracellular matrix production, cell growth, and cell differentiation that are not promoted by its function as a plasmin inhibitor. We herein investigated the role of α2AP in bone formation by examining calcein incorporation after its injection in α2AP-deficient mice. We found that α2AP deficiency enhanced the bone formation rate in mice. We also found that the osteocalcin expression and alkaline phosphatase activity were elevated in the femur and serum of the α2AP-deficient mice. Intriguingly, α2AP deficiency promoted osteoblast (OB) differentiation of primary calvarial OBs. In contrast, α2AP attenuated OB differentiation of mouse osteoblastic the MC3T3-E1 cells. Furthermore, α2AP attenuated Wnt-3a-induced β-catenin expression and low‑density lipoprotein receptor-related protein 6 activation in the MC3T3-E1 cells. These results suggest that α2AP negatively affects OB differentiation and function by inhibiting the Wnt/β-catenin pathway. These findings provide a basis for clinical strategies to improve various bone disorders.