Mesenchymal stromal cells (MSCs) have tremendous potential for use in regenerative medicine due to their multipotency and immune cell regulatory functions. Biomimetic physical forces have been shown to direct differentiation and maturation of MSCs in tissue engineering applications; however, the effect of force on immunomodulatory activity of MSCs has been largely overlooked. Here we show in human bone marrow-derived MSCs that wall shear stress (WSS) equivalent to the fluid frictional force present in the adult arterial vasculature significantly enhances expression of four genes that mediate MSC immune regulatory function, PTGS2, HMOX1, IL1RN, and TNFAIP6. Several mechanotransduction pathways are stimulated by WSS, including calcium ion (Ca2+) flux and activation of Akt, MAPK, and focal adhesion kinase (FAK). Inhibition of PI3K-Akt by LY294002 or Ca2+ signaling with chelators, ion channel inhibitors, or Ca2+ free culture conditions failed to attenuate WSS-induced COX2 expression. In contrast, the FAK inhibitor PF-562271 blocked COX2 induction, implicating focal adhesions as critical sensory components upstream of this key immunomodulatory factor. In co-culture assays, WSS preconditioning stimulates MSC anti-inflammatory activity to more potently suppress TNF-α production by activated immune cells, and this improved potency depended upon the ability of FAK to stimulate COX2 induction. Taken together, our data demonstrate that biomechanical force potentiates the reparative and regenerative properties of MSCs through a FAK signaling cascade and highlights the potential for innovative force-based approaches for enhancement in MSC therapeutic efficacy.