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Aim Osteoblasts are key functional cells in the process of bone metabolic balance. Phytoestrogens have an important influence on the proliferation and differentiation of osteoblasts. Puerarin, a plant estrogen, has a wide range concentration in vitro on the function of osteoblasts. The current study investigates the effect of the phytoestrogen puerarin on the proliferation, differentiation, and mineralization of osteoblasts in vitro . Methods The calvaria bone of eight-ten Wistar rats which were born within 24 h were obtained in aseptic condition. After enzyme digestion, isolation, purified osteoblasts of rats were cultured for further study. The cells of the first to third generation were divided into a control group and a puerarin-treated group with 10 −3 -10 −10 mol·L −1 puerarin. The cells were exposed to the medium containing a low level of carbohydrates, 10% ( V / V ) FBS for 24 h. After 1 to 4 days of culture, the OD values on the proliferation of osteoblasts in each group were determined by microplate reader. The cells were cultured in the medium containing 50 μg·mL −1 vitamin C, 10 −2 mol·L −1 sodium glycerophosphate, 10% FBS and the medium was changed every 3 to 4 days. After 2 to 8 days of culture, expression of alkaline phosphatase were tested and compared by microplate reader. The mineral nodes of osteoblasts were dyed using alizarin red or improved Von Kossa way after four weeks. Results Compared with those in the 10 −5 -10 −9 mol·L −1 puerarin, the proliferation of osteoblasts, the expression of alkaline phosphatase, and the number of mineral nodes of osteoblasts were significantly decreased in the control group. The increase was the fastest in the third day, while on the fourth day it was decreased, and arrived at statistical significance compared with the alkaline phosphatase activities and control group. The 10 −6 mol·L −1 group was the most distinct, and formed the most mineralized nodule. Compared with the 10 −3 mol·L −1 puerarin group, those changes were markedly increased in the control group. Conclusions Puerarin has proliferation, differentiation, and mineralization effects on osteoblasts in a dose-dependent manner, and has a double-way effect on the osteoblasts in vitro . A low-dose showed positive effects on the development of osteoblasts, and high-dose puerarin could inhibit the formation of bone.