In vivo functionality of cardiovascular tissue engineered constructs requires in vitro control of tissue development to obtain a well developed extracellular matrix (ECM). We hypothesize that ECM formation and maturation is stimulated by culturing at low oxygen concentrations. Gene expression levels of monolayers of human vascular-derived myofibroblasts, exposed to 7, 4, 2, 1, and 0.5% O2 (n = 9 per group) for 24 h, were measured for vascular endothelial growth factor (VEGF), procollagen 1(I) and 1(III), elastin, and cross-link enzymes lysyl oxidase (LOX) and lysyl hydroxylase 2 (LH2). After 4 days of exposure to 7, 2, and 0.5% O2 (n = 3 per group), protein synthesis was evaluated. All analyses were compared with control cultures at 21% O2. Human myofibroblasts turned to hypoxia-driven gene expression, indicated by VEGF expression, at oxygen concentrations of 4% and lower. Gene expression levels of procollagen 1(I) and 1(III) increased to 138 ± 26 and 143 ± 19%, respectively, for all oxygen concentrations below 4%. At 2% O2, LH2 and LOX gene expression levels were higher than control cultures (340 ± 53 and 136 ± 29%, respectively), and these levels increased even further with decreasing oxygen concentrations (611 ± 176 and 228 ± 45%, respectively, at 0.5% O2). Elastin gene expression levels remained unaffected. Collagen synthesis and LH2 protein levels increased at oxygen concentrations of 2% and lower. Oxygen concentrations below 4% induce enhanced ECM production by human myofibroblasts. Implementation of these results in cardiovascular tissue engineering approaches enables in vitro control of tissue development.