Angiogenesis, the growth of new capillaries from pre-existing ones, occurs through dynamic functions of the endothelial cells (EC), including migration, which is essential to achieve an organized formation of the vessel sprout. We demonstrated previously that an aspirin-triggered lipoxin analog, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A4 (ATL-1), inhibits vascular endothelial growth factor (VEGF)-induced EC migration. In the present study, we investigated the effects of ATL-1 in the actin cytoskeleton reorganization of EC stimulated with VEGF. Pretreatment of EC with ATL-1 caused a reduction in VEGF-induced stress fibers and therefore reduced the intracellular content of filamentous actin. A concomitant impairment in stress-activated protein kinase (SAPK2/p38) phosphorylation suggests that ATL inhibition of VEGF-stimulated actin polymerization involves the SAPK2/p38 pathway. Moreover, ATL-1 treatment inhibited focal adhesion clustering due to inhibition of focal adhesion kinase (FAK) phosphorylation and the subsequent association of FAK with the actin cytoskeleton. This final event, which ultimately allows cell migration, was reverted by an LX receptor antagonist, but not by a cys-LT1R antagonist, indicating an effect via the G-protein-linked LXA4 receptor. Together our results provide evidence that ATL-1 inhibits EC migration via the concerted inhibition of actin polymerization and proper assembly of focal adhesions, supporting a role for these novel lipid mediators as angiogenesis modulators.