The phospholipid-dependent protein kinase C is implicated in the regulation of cellular motility and energy metabolism. Phosphatidylserine, a main cofactor of protein kinase C, is involved in the regulation of glyceraldhehyde-3-phosphate dehydrogenase, which as actin, was shown to be phosphorylated by purified protein kinase C. Here, we study the effect of phosphatidylserine on the enzyme-substrate interaction of protein kinase C with glyceraldhehyde-3-phosphate dehydrogenase and actin. The stoichiometry of glyceraldhehyde-3-phosphate dehydrogenase phosphorylation is not affected by varying the level of phosphatidylserine. However, actin phosphorylation is dependent on phosphatidylserine level, peaking at high phosphatidylserine concentration. Moreover, if actin and glyceraldhehyde-3-phosphate dehydrogenase are cophosphorylated at high phosphatidylserine concentration, actin phosphorylation is favored, despite lower affinity for protein kinase C. Hence, phosphatidylserine directs differential phosphorylation of these key proteins of glycolysis and cellular motility and might be capable of recruiting protein kinase C for preferential actin phosphorylation. The sedimentation of phosphorylated actin is increased 3.8 fold and total actin 1.7 fold, suggesting that phosphorylation promotes actin polymerization.