Abstract: Objective　To explore the affects of actin on osteogenic differentiation of human fibroblast by regulating mitochondrial dynamics.　Methods 　Cytochalasin D (Cyto D) and Jasplakinolide (JAS) were added to be the growth medium and osteogenic differentiation medium respectively, then the changes of cell characters and mitochondrial dynamics were detected by Immunofluorescence staining and western blot experiments in the treatment of 1 d, 4 d and 7 d.　Results　Cyto D reduced the effect of osteogenic induction media, but JAS increased the effect of it. JAS enhanced significantly the effect of mitochondrial dynamics in fibroblast than Cyto D.　Conclusions　Actin polymerization motivates the osteogenic differentiation; Actin polymerization and depolymerization affects the colocalization ratio between mitochondrial and actin in osteogenic differentiated cells. The results can provide important biological information for studying the mitochondrial and differentiation mechanism.