MEKK1, which plays a major role in MAP kinase signaling, is implicated in mechanical functions of cells, such as migration. Here, we identify the actin-binding protein calponin-3 as a novel MEKK1 substrate in the signaling that regulates actomyosin-based cellular contractility. MEKK1 co-localizes with calponin-3 at the actin cytoskeleton and phosphorylates it, leading to an increase in the cell-generated traction stress. MEKK1-mediated calponin-3 phosphorylation is attenuated by the inhibition of myosin II activity, the disruption of actin cytoskeletal integrity, and the adhesion to soft extracellular substrates, whilst it is enhanced upon cell stretching. Our results reveal the significance of the MEKK1-calponin-3 signaling pathway in cell contractility.