Background/Aim: Protein phosphatase 1 (PP1) belongs to a major family of serine/threonine specific phosphatases and is universally expressed in all eukaryotic cells. PP1cβ is a specific isoform of the catalytic subunit of PP1 enzyme and its physiological activity contributes to the normal maintenance of some cellular functions such as endothelial cell barrier protection and DNA repair. We have previously showed that PP1cβ also plays a key role in endothelial cell angiogenesis. In particular, PP1cβ regulates the early stage of cell migration during the angiogenic process, rather than the subsequent phase of tube formation. Since previous studies have reported PP1cβ localization to focal adhesions, we hypothesized that PP1cβ regulation of endothelial cell migration would involve cytoskeletal reorganization through impairment of focal adhesion kinases (FAK) activation. Hence, the mechanism by which PP1cβ regulates endothelial cell angiogenesis was investigated in this study. Methods. The effect of PP1cβ pharmacological inhibition, in addition to knocking down and overexpressing PP1cβ, on endothelial cell migration and morphogenesis was examined using in vitro wound healing scratch assay. The PP1cβ knockdown effects on F-actin reorganization (phalloidin staining) and focal adhesion formation (vinculin) were evaluated by immunocytochemical staining with specific antibodies. Results: PP1cβ knockdown significantly reduced endothelial cell migration, with this effect being restored to the control level upon consecutive transfection with PP1cβ cDNA. The mechanism, by which PP1cβ regulates endothelial cell migration, involves the interplay of actin cytoskeleton proteins and focal adhesion molecules signalling. In particular, PP1cβ knockdown induced a profound cytoskeletal reorganization, loss of focal adhesion sites and impairment of focal adhesion kinases (FAK) activation. Conclusions: PP1cβ is regulator of endothelial cell migration, which is critical in the angiogenic process. PP1cβ inhibition reduces endothelial cell migration through focal adhesion turnover and actin polymerization pathways.