Abstract： Objective To investigate the effect of insulin-like growth factor- Ⅰ ( IGF- Ⅰ ) on the proliferation and osteogenesis of human periodontal ligament stem cells ( hPDLC ) under three-dimensional (3D) culture system. Methods Human periodontal cells were isolated from the ligament of surgically extracted human teeth, and through the limiting dilution assay, got mono-clone of the cell, hPDLCs were isolated from MesenPRO RS medium. Rotary cell culture system (RCCS) was enrolled to set 3D environment. Control group and experiment groups were assigned according to the concentration of IGF- Ⅰ .There were 5 level of experiment groups (0.1,1,10,50,100 μg/L). Proliferation was tested with methyl thiazolyl tetrazolium ( MTT), and alkine phosphatase (ALP) level was assayed by spectrophotometer to analyze the osteogenesis of hPDLCs. Gene expression of ostetocalcin(OCN)and type Ⅰ collagen (Col Ⅰ )were assayed by reverse transcriptase polymerase chain reaction(RT-PCR). Results In 3D culture system,the effect of IGF- Ⅰ on cell proliferation was significantly different between control group and experiment groups( P ＜ 0.05 ), and there showed significant differences between the group of 0.1 μg/L ( 0.219 ±0.021 ) IGF- Ⅰ and the groups of 50, 100 μg/L(0.287 ±0.011,0.293 ±0.012). However, there showed no significant differences among other groups. Significant differences of ALP activity were observed between the control group and experiment groups, and between the groups of 1, 10 μg/L(0.304 ±0.020, 0.310 ±0.013) and that of 50, 100 μg/L (0.347 ±0.011, 0.344 ±0.010) (P ＜0.05). While no significantdifferences were detected between the group of 1 μg/L and that of 10 μg/L, nor between the group of 50 μg/L and that of 100 μg/L. Expressions of Col Ⅰ and OCN in mRNA and protein level both showed dose-dependent increase. Conclusions In 3D culture system, in the scale of 0.1-100 μg/L, the effect of IGF-Ⅰ on the proliferation of hPDLCs increased dose-dependently. 100 μg/L IGF- Ⅰ promotes osteogenesis of the cells significantly.