OBJECTIVE: To evaluate the effect of platelet-rich fibrin extract (PRFe) on proliferation and differentiation and F-actin cytoskeleton of osteoblasts. METHODS: The experimental group used the α-minimum essential medium (α-MEM) containing PRFe (10% fetal bovine serum), and the control group used the α-MEM (10% fetal bovine serum). The number of the osteoblasts at 1st, 3rd, 5th d was detected by methyl thiazolyl tetrazolium (MTT) assay, and the differentiation of osteoblast at 1st, 3rd, 5th,7 th d detected by the activity of alkaline phosphatase (ALP).The alizarin red dye was used to observe the number of calcium nodus at 14th, 21st d. The F-actin cytoskeleton was evaluated by confocal laser scanning microscope(CLSM) at 3rd,6th,9th,12th h. The level of osteogenetic biomarkers osteocalcin (OCN) and core-binding factor α1(Cbfα1) at 3rd,7th d were quantified by real-time PCR. RESULTS: A significant increase of absorbance at 1st, 3rd, 5th d was showed in experimental group (0.336 ± 0.011, 0.571 ± 0.039, 0.787 ± 0.050) compared to control group (0.300 ± 0.021, 0.387 ± 0.040, 0.527 ± 0.034) (P < 0.05). The absorbance of experimental group at 1st, 3rd, 5th, 7th d (0.146 ± 0.014, 0.199 ± 0.017, 0.390 ± 0.020, 0.492 ± 0.019) was significantly higher than that of control group (0.115 ± 0.014, 0.145 ± 0.015, 0.190 ± 0.015, 0.230 ± 0.026) (P < 0.05). The integrated absorbance of the calcium nodus in experimental group at 14th, 21st d (22.119 ± 3.694, 31.528 ± 3.162) was significantly higher than in control group (8.498 ± 2.041, 15.162 ± 2.526) (P < 0.05). The Cbfα1 and OCN gene expression in experimental group was higher than in control group (P < 0.05). CONCLUSIONS: PRFe could enhance the proliferation and differentiation of osteoblasts and promote the spread of F-actin cytoskeleton.