Abstract: Objectives To investigate the time-dependent effects of serum from burned rats on cytoskeletal filamentous actin (F-actin) reorganization by visualizing their distribution in human umbilical vein endothelial cell line ECV-304 and evaluate the role of myosin light-chain kinase (MLCK) in this process. Methods The serum-starved ECV-304 cells were incubated with the serum from burned rats for 30 min, 1, 2, 4, and 6 h, respectively, and 30 min before or after the incubation, the cells were treated with 5 μmol/L ML-7 for 30 min. F-actin was stained with rhodamine-phalloidin and observed under fluorescence microscope. Results Under normal condition, F-actin was distributed mainly in the cortical area of the endothelial cells. After stimulation with the burn serum, stress fiber formation could be clearly seen in the endothelial cells, exhibiting a time-dependent enhancement in a time course ranging from 30 min to 6 h. Such an effect could be significantly inhibited by a 30-min pretreatment of the cells with MLCK-specific inhibitor ML-7. Inhibition of MLCK also reversed actin reorganization in the endothelial cells pretreated with the burn serum. Conclusion Serum from burned rats induces characteristic morphological changes in the endothelial cell actin cytoskeleton mainly due to the MLCK activation, an effect that can be reversed by the inhibition of MLCK.