Objective：To compare the effects of three root canal sealers with respect to time on biocompatibility of human periodontal ligament cells (hPDLCs).The sealers included zinc oxide and eugenol based sealers (ZOE), epoxy resin sealers (ERS) and silicone based sealers (SBS). Methods: hPDLCs were primarily cultured,with the method combining of tissue explant and enzymatic digestion. The cells were then exposed to different extract fluids：（1）ZOE extracted for 24 h group ；（2）ZOE extracted for 1 week group；（3）ZOE extracted for 2 weeks group；（4）ERS extracted after 24 h group；（5）ERS extracted after 1 week group；（6）ERS extracted after 2 weeks group；（7）SBS extracted after 24 h group；（8）SBS extracted after 1 week group；（9）SBS extracted after 2 weeks group；（10）Dulbecco modified Eagle’s medium/F12（DMEM/F12）as negative control group. Cell morphology was observed under an inverted microscope.Cell proliferation was measured by methyl-thiazol-diphenyltetrazolium （MTT）assay.ALP assay kit was used for measuring alkaline phosphatase (ALP) activity. Sealers of 2 weeks’ setting time were then immersed in an osteogenic medium for examination of mineral nodules and calcium deposits. Results: Considering the relative growth rate(RGR),ZOE was severely to moderately cytotoxic（RGR:13.6%-39.9%）,while ERS was slightly or not cytotoxic (RGR: 87.6%-95.3%).Only SBS did not show any cytotoxicity after setting (RGR: 91.8%-106.7%). The setting time influenced the cytotoxicity of ERS which decreased after 1 week. Considering the ALP activity,there was no difference between SBS group and control group（F=3.397，P=0.053）. According to the results of calcium deposits, ZOE：D562 nm= 0.180±0.050,ERS：D562 nm= 2.968±0.201,SBS：D562 nm= 3.623±0.039,Control：D562 nm= 3.477±0.102,the ranking of ALP activity and calcium deposits was as follows: ZOE