Bone metabolism has a close relationship with iron homeostasis. To examine the effects of iron excess and iron deficiency on the biological activities of osteoblast in vitro, human osteoblast cells (hFOB1.19) were incubated in a medium supplemented with 0-200 μmol/L ferric ammonium citrate and 0-20 μmol/L deferoxamine. The intracellular iron was measured by a confocal laser scanning microscope. Proliferation of osteoblasts was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptotic cells were detected using annexin intervention V/PI staining with a flow cytometry. Alkaline phosphatase (ALP) activity was measured using an ALP assay kit. The number of calcified nodules and mineral area was evaluated by von Kossa staining assay. The expressions of type I collagen and osteocalcin of cultured osteoblasts were detected by reverse transcriptase polymerase chain reaction and Western blot. Intracellular reactive oxygen species (ROS) was measured using the oxidation-sensitive dye 2,7-dichlorofluorescin diacetate by flow cytometry. The results indicated that excessive iron inhibited osteoblast activity in a concentration-dependent manner. Low iron concentrations, in contrast, produced a biphasic manner on osteoblasts: mild low iron promoted osteoblast activity, but serious low iron inhibited osteoblast activity. Osteogenesis was optimal in certain iron concentrations. The mechanism underlying biological activity invoked by excessive iron may be attributed to increased intracellular ROS levels.