Vimentin intermediate filaments undergo spatial reorganization in cultured smooth muscle cells in response to contractile activation; however, the role of vimentin in physiologic properties of smooth muscle has not been well elucidated. In this report, tracheal smooth muscle strips were loaded with antisense oligonucleotides against vimentin, and these muscle strips were then cultured for 2 days to allow for protein degradation. The treatment with vimentin antisense but not sense oligonucleotides suppressed vimentin protein expression; neither antisense nor sense oligonucleotides affected protein levels of desmin and actin. Force development in response to stimulation with acetylcholine (ACh) or KCl depolarization was lower in vimentin-deficient tissues than in muscle tissues treated with sense oligonucleotides or in muscle strips not treated with oligonucleotides. Passive tension was also depressed in vimentin-depleted muscle tissues. Vimentin downregulation did not attenuate increases in myosin light chain phosphorylation in response to contractile stimulation or basal myosin light chain phosphorylation. In smooth muscle strips that had been treated with vimentin sense or without oligonucleotides, the desmosomal protein plakoglobin was primarily localized in cell periphery. The membrane-associated localization of plakoglobin was reduced in vimentin-depleted muscle tissues. These studies suggest that vimentin filaments play an important role in mediating active force development and passive tension, which is not regulated by myosin light chain phosphorylation. Vimentin downregulation impairs the structural organization of desmosomes, which may be associated with the decrease in force development.