Dynamic cytoskeletal changes appear to be one of intracellular signals that control cell differentiation. To test this hypothesis, we examined the effects of short-term actin cytoskeletal changes on osteoblastic differentiation. We found an actin polymerization interfering reagent, cytochalasin D, promoted osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells. We also found that these effects were mediated by the protein kinase D (PKD) pathway. Short-term cytochalasin D treatment increased alkaline phosphatase (ALP) activity, osteocalcin (OCN) secretion, and mineralization of the extracellular matrix in MC3T3-E1 cells, with temporary changes in actin cytoskeleton. Furthermore, the disruption of actin cytoskeleton induced phosphorylation of 744/748 serine within the activation loop of PKD in a dose-dependent manner. The protein kinase C (PKC)/PKD inhibitor Go6976 suppressed cytochalasin D-induced acceleration of osteoblastic differentiation, whereas Go6983, a specific inhibitor of conventional PKCs, did not. Involvement of PKD signaling was confirmed by using small interfering RNA to knock down PKD. In addition, another actin polymerization interfering reagent, latrunculin B, also stimulated ALP activity and OCN secretion with PKD activation. On the other hand, the present data suggested that transient dynamic actin cytoskeletal reorganization could be a novel cellular signal that directly stimulated osteoblastic differentiation.