Objective To investigate the effect of fluid shear stress (FSS) on p21 expression in osteoblasts, and to clarify the role of p21 in osteoblast proliferation induced by FSS. Methods Osteoblasts were treated with 1. 2 Pa FSS for different duration ( 0, 15, 30, 45, 60 min). Osteoblasts were transfected with siRNAp21 or pcDNAp21, and the transfection effect was detected by Western blotting. The proliferation of osteoblasts was assessed by CCK-8 assay or EdU labelling assay. The expression of p21, cyclinD1 and CDK4 was detected by Western blotting. Results 1. 2 Pa FSS significantly down-regulated p21 expression, and the expression level of p21 was the lowest when treated with FSS for 45 min. Both FSS and down-regulation of p21 significantly increased the proliferation of osteoblasts and the expression of cyclin D1 and CDK4. However, after up-regulating p21 expression, FSS couldn’ t increase the proliferation of osteoblasts and the expression of cyclin D1 and CDK4. Conclusions 1. 2 Pa FSS can down-regulate p21 expression in osteoblasts, and the expression level of p21 was the lowest when treated with FSS for 45 min. Down-regulation of p21 can promote osteoblast proliferation, and FSS promotes osteoblast proliferation through down-regulating p21.