Macrophages are essential for inflammation, tissue repair, and tissue homeostasis. Dysregulated macrophage function results in chronic inflammation that underlies major diseases including cardiovascular disease, cancer, and fibrosis. Chronic inflammation causes excessive remodeling of extracellular matrix that increases elasticity, or stiffness, of tissues. Cells sense and respond to changes in extracellular elasticity by mechanotransduction pathways mediated by integrins and downstream mediators (e.g. focal adhesion kinase, rho-associated coiled coil kinase), ion channels (e.g. TRP and PIEZO channels), and transcription factors. At the beginning of this project in 2013, few studies had investigated mechanoregulation macrophage function, and even fewer had investigated the mechanoregulation of Toll-like receptor (TLR) signaling. My studies described herein led to three major advances in our understanding of mechanoregulation of macrophages. In chapter 2, I demonstrated that Toll-like receptor signaling in macrophages is regulated by extracellular substrate stiffness and Rho-associated coiled-coil kinase (ROCK1/2). I modeled physiologically- and disease-relevant differences in substrate elasticity using tunable polyacrylamide gels, and showed that substrate elasticity and ROCK1/2 signaling inhibit TLR signaling and decrease cytokine secretion. These studies suggest that modulating stiffness or macrophage response to stiffness is a potential mechanism to treat chronic inflammation. In chapter 3, I demonstrated that macrophage uptake of oxidized and acetylated low-density lipoproteins and generation of reactive oxygen species are regulated by stiffness of the growth surface. These studies have high relevance to atherosclerosis, which is associated with profound changes in vessel wall stiffness, and suggest that increased vessel wall stiffness may directly increase macrophage uptake of lipid and contribute to pathophysiology. In chapter 4, I demonstrated that transient receptor potential vanilloid 4 (TRPV4) is required for maximal TLR-mediated cytokine release. These findings expand on studies published during my dissertation research that showed that TRPV4 mediates lipopolysaccharide-induced phagocytosis. Intriguingly, I also provide preliminary evidence that a widely used TRPV4-/- mouse model may express a TRPV4 splice variant that does not mediate calcium flux, but still promotes TLR signaling. My findings could help the development and rational use of emerging therapies that decrease tissue elasticity or cellular response to increased elasticity. Capitalizing on mechanotransduction in macrophages could be used to halt or attenuate the chronic inflammation that underlies a multitude of the most pressing chronic diseases.