All cell functions that involve membrane deformation or a change in cell shape (e.g., endocytosis, exocytosis, cell motility, and cytokinesis) are regulated by membrane tension. While molecular contacts between the plasma membrane and the underlying actin cytoskeleton are known to make significant contributions to membrane tension, little is known about the molecules that mediate these interactions. We used an optical trap to directly probe the molecular determinants of membrane tension in isolated organelles and in living cells. Here, we show that class I myosins, a family of membrane-binding, actin-based motor proteins, mediate membrane/cytoskeleton adhesion and thus, make major contributions to membrane tension. These studies show that class I myosins directly control the mechanical properties of the cell membrane; they also position these motor proteins as master regulators of cellular events involving membrane deformation.