This study sought to assess the effect of a new nano-root filling material, nano-hydroxyapatite polyamide66 (nHA-PA66), on the growth of osteoblast cultured in vitro so as to evaluate its osteoblast-compatibility and potential to be used as root canal filling material. The Dulbecco's Modified Eagle Media (DMEM) leaching liquor of nHA-PA66 was applied to the osteoblasts of the test group while the DMEM itself was applied to control. The MTT assay and flow cytometry were used to evaluate the changes in cell growth and cell cycle. The relative proliferative ratio of different concentrations of nHA-PA66 ranged between 98%-106% without dose-dependent effect. The proportion and time of different subcycle of the two groups were close to each other. nHA-PA66 showed no negative effects on the osteoblasts' growth and cell cycle. These led to the suggestion that nHA-PA66 is not cytotoxic to osteoblast and is a promising material in clinical use.