We previously showed that P4-ATPases, ATP10A/ATP8B1, and ATP11A/ATP11C have flippase activities toward phosphatidylcholine (PC), and aminophospholipids [phosphatidylserine (PS) and phosphatidylethanolamine], respectively. Here, we investigate the effect of PC-specific flippases versus aminophospholipid-specific flippases in cell spreading on the extracellular matrix. Expression of PC-flippases, but not PS-flippases, delayed cell adhesion, cell spreading and inhibited formation of focal adhesions. In addition, overexpression of a PS-binding probe that sequesters PS in the cytoplasmic leaflet delayed cell spreading and inhibited formation of focal adhesions. These results suggest that elevation of PC at the cytoplasmic leaflet of the plasma membrane by expression of PC-flippases may reduce the local concentration of PS or phosphoinositides, required for efficient cell adhesion, focal adhesion formation, and cell spreading.