Plant cells are encased in cell walls which are important for the growth and development of the organism. Primary cell wall consists mainly of polysaccharides with cellulose as the most abundant component. In plant cells, cellulose is synthesized by a plasma membrane (PM) localized protein complex called the cellulose synthase complex (CSC). It was previously reported that disrupting normal actin organization resulted in a reduction of cellulose content in Arabidopsis dark-grown seedlings. Furthermore, actin was found to facilitate the delivery of CSC into the PM, and inferred to be involved in endocytosis. As a motor protein that translocates cargo along actin filaments, myosin plays an important role in organelle and vesicle trafficking. However, it is not known whether myosin is involved in regulating cellulose deposition or CSC behavior. Here, we used biochemical analysis to determine the cellulose content in Arabidopsis etiolated seedlings, and found a significantly decreased cellulose content in a myosin xi-1, xi-2, and xi-k triple knockout mutant (xi3KO), indicating that myosin is involved in cellulose deposition. To evaluate the molecular mechanism underlying the role of myosin in CSC trafficking, we characterized and employed a new plant myosin inhibitor