Cellular traction forces are known to play an important role in the interactions between cells and their substrates. Although several studies have estimated traction forces using microfabrication techniques, little is known of how intracellular structures contribute to traction forces. In this study, microfabricated substrates with different arrays of microposts were used to estimate traction forces of smooth muscle cells, particularly exploring the contribution of cytoskeletal structures to traction forces. Smooth muscle cells isolated from bovine aortas were transfected with fluorescent protein conjugates, and then plated on an elastomer substrate with arrays of microposts (3 μm in diameter, 10 μm in height, 6 or 10 μm in spacing). The total area of the microposts arrays was designed to be 30 μm x 30 μm to control cell area. A BrdU incorporation assay revealed that cell proliferation significantly decreased when the spacing of microposts was increased from 6 to 10 μm. A significant increase in traction forces from 15.2 ± 1.4 to 22.4 ± 1.8 nN was also found by increasing the spacing of the microposts, although less organization of stress fibers was observed. This result indicates that, for the 10 μm spacing, actin-myosin interactions may be more activated, producing higher forces per microposts. Moreover, when microtubules were disrupted, using 10 μg/ml nocodazole, a 30% increase in traction forces was observed in cells plated on both sets of microposts. Therefore, this study proposes that the contraction forces of stress fibers are mainly involved in cellular traction forces, and that microtubules, directly or indirectly, contribute to the contraction forces of stress fibers.