Phenotypic polarization of macrophages is deemed essential in innate immunity and various pathophysiological processes, but little is known about how mechanical forces like matrix stiffness regulate the polarization and the associated signaling mechanisms. Here we report that calcium ion channel Transient Receptor Potential Vanilloid 4 (TRPV4), a mechanosensitive receptor/channel, mediates the substrate stiffness-induced macrophage polarization. Using atomic force microscopy, we show that fibrosis-induced tissue stiffness is dependent on TRPV4. M1 macrophages were found to be the predominant macrophage subtype in stiffer tissues and loss of TRPV4 significantly decreased the level of M1 macrophages. These findings were further validated by our in vitro assays indicating that increase in substrate stiffness leads to an increased secretion of M1 proinflammatory mediators, which is further enhanced by the addition of soluble factors. Taken together, these findings provide new insights about the role of TRPV4 in matrix stiffness-induced macrophage polarization that can be explored in tissue engineering and in the development of targeted therapeutics.