Airway inflammation is a hallmark of asthma triggering airway smooth muscle (ASM) hyperreactivity and airway remodeling. TNFα increases both agonist-induced cytosolic Ca²⁺ concentration and force in ASM. The effects of TNFα on ASM force may also be due to an increase in Ca²⁺ sensitivity, cytoskeletal remodeling and/or changes in contractile protein content. We hypothesized that 24-h exposure TNFα increases ASM force by changing actin and myosin heavy chain (MyHC) content and/or polymerization. Porcine ASM strips were permeabilized with 10% Triton X-100, and force was measured in response to increasing concentrations of Ca²⁺ (pCa 9.0 to pCa 4.0) in control and TNFα treated groups. Relative phosphorylation of the regulatory myosin light chain, total actin, MLC and myosin heavy chain (MyHC) concentrations were quantified at pCa 9.0, pCa 6.1 and pCa 4.0. Actin polymerization was quantified by the ratio of filamentous to globular actin at pCa 9.0 and pCa 4.0. Total cross-bridge formation was determined by measuring isometric ATP hydrolysis rate at pCa 4.0 using an enzyme-coupled NADH-fluorometric technique. Exposure to TNFα significantly increased force across the range of Ca²⁺ activation, but did not affect the intrinsic Ca²⁺ sensitivity of force generation. The TNFα-induced increase in ASM force was associated with an increase in total actin, MLC and MyHC content as well as increased actin polymerization and increase in maximum isometric ATP hydrolysis rate. The results of this study support our hypothesis that TNFα increases force generation in ASM by increasing the number of contractile units (actin-myosin content) contributing to force generation.