Mechanical stimulation of osteoblasts activates many cellular mechanisms including the release of ATP. Binding of ATP to purinergic receptors is key to load-induced osteogenesis. Osteoblasts also respond to fluid shear stress (FSS) with increased actin stress fiber formation (ASFF) that we postulate is in response to activation of the P2Y₂ receptor (P2Y₂R). Further, we predict that ASFF increases cell stiffness and reduces the sensitivity to further mechanical stimulation. We found that siRNA suppression of P2Y₂R attenuated ASFF in response to FSS and ATP treatment. In addition, RhoA GTPase was activated within 15 minutes after the onset of FSS or ATP treatment and mediated ASFF following P2Y₂R activation via the ROCK1/LIM Kinase 2/cofilin pathway. We also observed that ASFF in response to FSS or ATP treatment increased the cell stiffness and was prevented by knocking down P2Y₂R. Finally, we confirmed that the enhanced cell stiffness and ASFF in response to RhoA GTPase activation during FSS drastically reduced the mechanosensitivity of the osteoblasts based on the [Ca²⁺]_i response to consecutive bouts of FSS. These data suggest that osteoblasts can regulate their mechanosensitivity to continued load through P2Y₂R activation of the RhoA GTPase signaling cascade, leading to ASFF and increased cell stiffness.