Abstract: OBJECTIVE To study the role of primary cilium in the osteogenic differentiation process of rat calvarial osteoblasts induced by osthole. METHODS The neonatal SD rat skull was segregated, and enzyme digestion was used to obtain bone cells. Primary cilium of osteoblasts was detected by immunostaining and cilium was abrogated by knockdown of IFT88. Transfected cells were treated with 1×10^-6 mol·L^-1 osthole. The alkaline phosphatase (ALP) activity was determined at the 3rd and 6th day. Total RNA was isolated and the gene expression of ALP, collagen-1(COL-1) and RUNX-2 were investigated by real-time PCR. Finally total protein was also isolated and the secretion of COL-1 and RUNX-2 was examined by Western blotting. RESULTS The percentage of cells possessing primary cilia was found to be >70%, and the cilium was about 5 mm in length projecting from cell surface. Knockdown of IFT88 caused an reduction of cells with primary cilia. The ALP activity of the group treated with 1×10^-6?mol·L^-1 osthole was significantly higher than the control group, and mRNA expression levels of ALP, COL-1 and RUNX-2 after of treatment changed in similar tendency. Similarly, the stimulating effects of osthole on protein expression of COL-1 and RUNX-2. However, osthole-induced promoting effects on ALP activity and other osteogenesis-related markers were suppressed when primary cilia of osteoblasts were abrogated using RNA interference. CONCLUSION The abrogation of primary cilium can dramatically blocked the differentiation and mineralization of osteoblasts induced by osthole.