This research examines the role of actin cytoskeleton filaments in chondroinduction by cyclic hydrostatic pressurization. A chondroinductive hydrostatic pressurization system was developed and characterized. A pressure of 5 MPa at 1 Hz frequency, applied for 7200 cycles (4 hours intermittent) per day, induced chondrogenic differentiation in C3H10T1/2 cells while 1800 cycles (1 hour intermittent) did not induce chondrogenesis. Quantitative analysis of chondrogenesis was determined as sulfated glycosaminoglycan synthesis and rate of collagen synthesis while qualitative analysis was obtained as Alcian Blue staining and collagen type II immunostaining. Actin disruption using 2 uM Cytochalasin D inhibited the enhanced sGAG synthesis in the chondroinductive hydrostatic pressurization environment and significantly inhibited rate of collagen synthesis to the mean level lower than that of the non-pressurized group. These results suggest an involvement of actin cytoskeleton filaments in mechanotransduction of cyclic hydrostatic pressure.