Objective To investigate the effect of focal adhesion kinases (FAK) inhibitor with different concentration on the adhesion and migration of endothelial cells (ECs) and the expression of downstream Rac1 protein, and to explore the role of FAK in adhesion and migration of ECs by using FAK inhibitor to inhibit the phosphorylation of Y397 site of FAK. Method Scratch wound migration assay was performed to examine the effect of FAK inhibitor with different concentration (from 0 nmol/mL to 250 nmol/mL) on ECs migration at 2, 4, 8 and 24 h, respectively. Western blot combined with immunofluorescence analysis were performed to determine the effect of FAK inhibitor with different concentration on distribution and expression of Rac1 protein. Results With the concentration of FAK inhibitor increased, ECs migration distance and the Rac1 protein expression decreased. Conclusions The inhibition of FAK phosphorylation could inhibit cell adhesion and migration with the decrease in downstream Rac1 protein, and ECs adhesion/migration was related to FAK Rho GTPases signaling pathways.