Physical force environment is a major factor that influences cellular homeostasis and remodelling. It is not well understood, however, as a potential role of force intensities in the induction of cellular mechanotransduction. Using a fluorescence resonance energy transfer-based approach, we asked whether activities of GTPase RhoA in chondrocytes are dependent on intensities of flow-induced shear stress. We hypothesized that RhoA activities can be either elevated or reduced by selecting different levels of shear-stress intensities. The result indicates that C28/I2 chondrocytes have increased RhoA activities in response to high shear stress (10 or 20 dyn/cm2), whereas a decrease in activity was seen with an intermediate shear stress of 5 dyn/cm2. No changes were seen under low shear stress (2 dyn/cm2). The observed two-level switch of RhoA activities is closely linked to the shear-stress-induced alterations in actin cytoskeleton and traction forces. In the presence of constitutively active RhoA (RhoA-V14), intermediate shear stress suppressed RhoA activities, while high shear stress failed to activate them. In chondrocytes, expression of various metalloproteinases is, in part, regulated by shear and normal stresses through a network of GTPases. Collectively, the data suggest that intensities of shear stress are critical in differential activation and inhibition of RhoA activities in chondrocytes.