Stargazin is the first transmembrane protein known to associate with AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate) glutamate receptors (AMPARs) and regulate their synaptic targeting by two distinct mechanisms, specifically via delivery of AMPARs to the surface membrane and synaptic targeting of these receptors by binding to PSD-95/SAP-90 and related PDZ proteins. However, it is not known whether and how this stargazin-mediated synaptic targeting of AMPARs is regulated. Stargazin interacts with the PDZ domains of PSD-95 through the C-terminal PDZ-binding motif. The stargazin C terminus contains a consensus sequence for phosphorylation by cAMP-dependent protein kinase A (PKA). Phosphorylation site-specific stargazin antibodies reveal that the stargazin C terminus is phosphorylated at the Thr-321 residue in heterologous cells and in vivo. Stargazin phosphorylation is enhanced by the catalytic subunit of PKA. Mutations mimicking stargazin phosphorylation (T321E and T321D) lead to elimination of yeast two-hybrid interactions, in vitrocoimmunoprecipitation, and coclustering between stargazin and PSD-95. Phosphorylated stargazin shows a selective loss of coimmunoprecipitation with PSD-95 in heterologous cells and limited enrichment in postsynaptic density fractions of rat brain. These results suggest that phosphorylation of the stargazin C terminus by PKA regulates its interaction with PSD-95 and synaptic targeting of AMPARs.