Ischemic wounds are characterized by oxygen levels lower than that of healthy skin ( hypoxia) and poor healing. To better understand the pathophysiology of impaired wound healing, we investigated how switching from high ( 21%) to low ( 2%) oxygen levels directly affects cultured skin myofibroblasts, essential cells for the normal wound repair process. Myofibroblast differentiation and function were assessed by quantifying alpha-smooth muscle actin expression and cell contraction in collagen gels and on wrinkling silicone substrates. Culture for 5 days at 2% oxygen is perceived as hypoxia and significantly reduced myofibroblast differentiation and contraction despite high levels of the profibrotic transforming growth factor-beta 1. Analysis of alpha-smooth muscle actin expression on wrinkling substrates over time showed that reduced myofibroblast contraction preceded alpha-smooth muscle actin disassembly from stress fibers after switching from 21 to 2% oxygen. These effects were reversible by restoring high oxygen conditions and by applying mechanical stress. We suggest that mechanical challenge is a clinical relevant strategy to improve ischemic and chronic wound healing by supporting myofibroblast formation.