Background: Inflammation is involved in the pathogenesis of calcific aortic valve stenosis, and aortic valve interstitial cells (AVICs) play an important role in valve disease and remodeling. We have previously shown that human AVICs express functional toll-like receptor 4 (TLR4), and express increased levels of ICAM-1 in response to stimulation with bacterial lipopolysaccharide (LPS). This study examines (1) the role of TLR4 in regulating ICAM-1 expression in human AVICs, (2) the interaction of ICAM-1 with microfilaments, and (3) the influence of microfilament integrity on ICAM-1 expression. Materials and Methods: AVICs isolated from explanted human hearts were stimulated with LPS (0.2μg/mL) to induce ICAM-1 expression, as shown by Western blot. TLR4 activity was influenced with a neutralizing antibody or by gene knockdown with siRNA. Flow cytometry and immunofluorescence were used to characterize membrane localization of ICAM-1. Immunoprecipitation and immunofluorescence were employed to show colocalization of ICAM-1 with microfilaments. Microfilament function was influenced with an actin polymerization inhibitor as well as siRNA. Results: Cellular ICAM-1 levels increased 10-fold after 24h of LPS stimulation, and this increase was significantly attenuated by prior treatment with TLR4-neutralizing antibody or TLR4 siRNA. ICAM-1 exhibited a filamentous distribution pattern after 4h LPS stimulation, which was associated with increased total and cell-surface ICAM-1 expression. ICAM-1 colocalized and coprecipitated with the microfilament proteins F-actin and α-actinin 1. Pretreatment with the actin polymerization inhibitor latrunculin B and silencing α-actinin 1 with siRNA reduced LPS-induced total and cell-surface ICAM-1 expression. Conclusions: This study demonstrates that (1) LPS-stimulated ICAM-1 expression in human AVICs is mediated by TLR4, (2) ICAM-1 interacts with microfilaments soon after LPS stimulation, and (3) microfilament disruption reduces ICAM-1 expression. These results suggest that the interaction between ICAM-1 and microfilaments facilitates LPS-induced ICAM-1 expression in human AVICs. © 2011 Published by Elsevier Inc. All rights reserved.