Spinophilin is a protein that binds to protein phosphatase-1 and actin and modulates excitatory synaptic transmission and dendritic spine morphology. We have identified three sites phosphorylated by ERK2 (Ser-15 and Ser-205) and cyclin-dependent PK 5 (Cdk5) (Ser-17), within the actin-binding domain of spinophilin. Cdk5 and ERK2 both phosphorylated spinophilin in intact cells. However, in vitro, phosphorylation by ERK2, but not by Cdk5, was able to modulate the ability of spinophilin to bind to and bundle actin filaments. In neurons and HEK293 cells expressing GFP-tagged variants of spinophilin, imaging studies demonstrated that introduction of a phospho-site mimic (Ser-15 to glutamate) was associated with increased filopodial density. These results support a role for spinophilin phosphorylation by ERK2 in the regulation of spine morphogenesis.