Objective To investigate the effect of insulin-like growth factor-Ⅰ(IGF-Ⅰ) on the proliferation and alkaline phosphatase(ALP) activity of human periodontal ligament cells(hPDLCs) under three-dimensional(3D) culture system. Methods The hPDLCs were cultured from periodontium of human teeth by the outgrowth method. Rotary cell culture system(RCCS) was enrolled to set 3D culture system. Samples were set to four groups: Negative control group, positive control group(3D group, IGF-Ⅰ group), and experimental group(3D with IGF-Ⅰ group). Proliferation was tested with methylthiazolyl tetrazolium(MTT), and ALP activity was assayed by spectrophotometer at 1, 3, 5, 7 d respectively. Results Compared with that of negative control group, cell proliferation increased significantly in 3D with IGF-Ⅰ group since 3 d(P