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Mesenchymal stem cells (MSCs) can maintain immune homeostasis and many preclinical trials with MSCs have been carried out around the world. In vitro culture of MSCs has been found to result in the decline of immunomodulatory capacity, migration and proliferation. To address these problems, simulating the extracellular environment for preconditioning of MSCs is a promising and inexpensive method. Biophysical cues in the external environment that MSCs are exposed to have been shown to affect MSC migration, residency, differentiation, secretion, etc. We review the main ways in which MSCs exert their immunomodulatory ability, and summarize recent advances in mechanical preconditioning of MSCs to enhance immunomodulatory capacity and related mechanical signal sensing and transduction mechanisms.