Actin microfilaments (F-actin) serve as the track for directional movement of organelles in plant cells. In actively growing plant cells, F-actin often form robust bundles that trespass the cellular dimension. To test how the F-actin network was employed for peroxisome movement, we wished to disturb actin organization by genetically compromising the function of villin (VLN) proteins that serve as the primary bundling factor in Arabidopsis thaliana cells. To do so, we isolated T-DNA insertional mutants in three VLN genes that were most actively expressed in vegetative tissues. We found that the vln4 mutation greatly enhanced the growth defects caused by the vln2 vln3 double mutant as the vln2 vln3 vln4 triple mutant had a great reduction of organ growth and formed heavily deformed tissues. Both VLN2 and VLN4 proteins were detected on bundled F-actin filaments. Compared to the wild-type cells, the double and triple mutants exhibited progressively reduction of stable F-actin bundles and had fine F-actin filaments undergo rapid remodeling. The defective F-actin network did not prevent peroxisomes from taking on both rapid and slow movements along the F-actin tracks. However, we found that compromised F-actin bundling caused significant reductions in the speed of peroxisome movement and the displacement distance of peroxisome positions. Using a correlation analysis method, we also demonstrated that the complex heterogeneous peroxisome movement may be classified into clusters reflecting the directionality of peroxisome movement. The triple mutant suffered from a significant reduction of peroxisomes exhibiting long-range and linear movement. Our results provided insights into how VLN-dependent F-actin organization was coupled with the complex patterns of peroxisome movement.