In order to study the effect of mechanical strain on mesenchymal stem cells (MSCs) differentiation into osteogenic, the cyclic substrate deformation instructment was applicated to MSCs line (D1 cell) in osteogenic media for different strain and times. The cells were cyclically stretched for periods of 1, 2, 3, 5 and 10 min and treated with cytochalasin B(CB) and EGTA. Ca²⁺, which were loaded with (15 μl) Fluo-3 AM , were detected by confocal laser scanning microscope(CLSM). The signaling inhibitors, such as SB203580(p38MAPK specific inhibitor), PD98059(MEK-1/2MAPK specific inhibitor), LY294002(PI3Ks specific inhibitor), cytochalasin B(microfilament specific inhibitor), EGTA (Ca²⁺ specific inhibitor), were used to investigate mechanical signaling pathway by (3% 0.5Hz) Cyclic strain. The results indicated that 3% strain stimulus could induce increasement of [Ca²⁺]_i - dependent fluorescence at 2 min. Its fluorescence intensity were 143.68,which were significantly different compared with 1,3,5,10min groups and 1% and 10% strain groups (P < 0.01). The increasement of Ca²⁺ levels were delayed upto 10 min, when the cells were treated with CB. EGTA (5 mmol/L) could inhibited strain induction increasment of Ca²⁺ levels. Strain (3%, 0.5 Hz) could induce Ca²⁺ spark event in D1 cells at 2 min, however the cells were treated with CB Ca²⁺ spark event were delayed until 5min. The expression of OCN and OSX mRNA were completely inhibited by five specific inhabitors in unstrained condition. Inhibition of ERK1/2 and p38 pathway promoted the expression of OSX and OCN mRNA by strain.The expression of OSX and OCN mRNA to inhibit PI₃Ks pathway were partly activated by strain. Application of strain could not activate OSX and OCN mRNA expression when extracellular Ca²⁺ greatly inhibited with EGTA (5 mmol/L) and microfilament were damaged with CB.These results demonstrate that mechanical signals regulate MSCs function, suggested Ca²⁺ signaling, PI₃Ks pathway and microfilament play an important role in transduction mechanical signal in MSCs differentiation.