Hydroxyapatite (HA) has been widely used as artificial bone substitutes because of its good biological activity, but the disadvantages of pure HA have limited its clinical applications. In human natural hard tissues, there are many trace elements doped in hydroxyapatite, which play beneficial roles in biological actions. In this study, multi-doped nanostructured hydroxyapatite (HA(2)) was synthesized by doping trace elements in HA according to the contents in human bones. Alkaline phosphatase activity staining, semi-quantitative PCR measurement (ALP, OCN and OPN) and alizarin red staining were carried out to detect the effect of pure nanostructured hydroxyapatite (HA(1)) and multi-doped nanostructured hydroxyapatite (HA(2)) on proliferation and differentiation of MC3T3-E1cells. Alkaline phosphatase activity experiments prove that the HA(2) has a stronger role than HA(1) in promoting cell production. Semi quantitative PCR measurement confirms that HA(2) is more effective than HA(1) in promoting the expression of osteogenic genes. Alizarin red staining results show that HA(2) is better than HA(1) for MC3T3-E1 cells to produce mineralized nodules. Multiple-doped nanostructured hydroxyapatite (HA(2)) has an obvious promoting effect on the differentiation and osteogenesis of MC3T3-E1 comparing with the undoped nanostructured HA(1). Multi-doped nanostructured hydroxyapatite (HA(2)) is more beneficial to bone formation, which has the inorganic composition more close to human bone and is a promising biomaterial for human hard tissue implants.