AIM: To evaluate the effect of endothelial myosin light chain kinase on extravasation migration of sarcoma cell. METHODS: An in vitro model of sarcoma cell transmigration across a monolayer of HUVEC cultured on collagen gel was used to observe extravasation migration of sarcoma cell and calculated the electrical resistance of HUVEC monolayer in extravasation migration of sarcoma cell. RESULTS: Sarcoma cell migrated through a gap between adjacent endothelial cells into collagen gel. The electrical resistance of a HUVEC monolayer reduced in extravasation migration of sarcoma cell.Endothelial myosin light chain kinase inhibitor(ML-7) inhibited extravasation migration of sarcoma cell and inhibited reduction of electrical resistance of a HUVEC monolayer in extravasation migration of sarcoma cell in a dose-dependent manner. CONCLUSION: Endothelial myosin light chain kinase regulates sarcoma cell transendothelial migration by phosphorylation of myosin light chain.