The study was aimed to investigate the role of the "extracellular matrix (ECM)-integrins-cytoskeleton" signal pathway in mechanotransduction of centrifugal loading. MG-63 osteoblasts were exposed to centrifugal loading at 209xg for 10 min. Uncentrifuged cells and centrifuged cells that have been trypsinized and suspended in liquors were designed as control. The changes in F-actin and alpha-actin cytoskeleton, gene transcription of ECM components, and integrins expression were analyzed by LSCM, Real-Time RT-PCR and FCM, respectively. A temporary and fast reversible change was observed in F-actin and alpha-actin cytoskeleton. And the change was paralleled with the fast autoregulation in gene transcription of ECM components of fibronection, osteopontin and Collagen I, and integrins expression of both alpha2 and beta1 subunits. The result suggested that cytoskeleton was a possible mechanical sensor to centrifugal stimuli, and the cytoskeleton regulation to centrifugal loading was in an ECM-dependent and integrin-mediated manner.