Primary cilia are responsible for sensing mechanical loading in osteocytes. However, the underlying working mechanism of cilia remains elusive. An osteocyte model is necessary to reveal the role of cilia. Furthermore, the osteocyte model should be with upregulated or downregulated primary cilium expression. Herein, we used a pharmacological method to regulate the cilium formation of osteocytes. After screening, some pharmacological agents can regulate the cilium formation of osteocytes. We performed a CCK-8 assay to analyze the optimal working conditions of the drugs for MLO-Y4 cells. The agents include chloral hydrate (CH), Gd³⁺+, Li⁺+, and rapamycin. The expression of cilia affects the cellular functions, including mechanosensitivity, of osteocytes. Results showed that CH downregulated the cilium formation and ciliogenesis of osteocytes. In addition, Gd³⁺+, Li⁺+, and rapamycin upregulated the cilium expression of osteocytes. Moreover, the cilium expression positively correlated with the mechanosensitivity of osteocytes. This work reveals the role of primary cilia in the mechanosensing of osteocytes.