AMPA-type glutamate receptors (AMPARs) play a critical role in mediating the majority of fast excitatory synaptic transmission in the brain, where alterations in receptor expression, distribution and trafficking have been shown to underlie synaptic plasticity and higher brain function. However, the molecular mechanisms regulating AMPAR surface expression and turnover are still not fully understood. We report that mammalian AMPARs are subject to post-translational modification by ubiquitin, and identify Nedd4 as the E3 ligase responsible for mediating this process. AMPAR ubiquitination enhanced receptor degradation and reduced AMPAR cell-surface expression; conversely, inhibition of proteasomal activity caused AMPAR accumulation. Using site-directed mutagenesis we replaced each of four lysine residues available as putative ubiquitination sites on the AMPAR subunit GluA1 C-terminal with an arginine and identified critical residues for ubiquitination and receptor degradation. Consistent with the role of protein ubiquitination, lysine mutation reduced the efficiency of AMPAR endocytosis. We further investigated the molecular mechanisms involved in the internalization of ubiquitinated AMPARs. We find that the endocytic adaptor protein Eps15 plays a critical role in this process. siRNA-mediated suppression or overexpression of Eps15 results in changes in AMPAR surface expression. Eps15 interaction with AMPARs requires Nedd4-mediated GluA1 ubiquitination along with the ubiquitin interacting motif (UIM) of Eps15. Consistent with ubiquitination-mediated receptor internalization, knockdown of Eps15 suppresses GluA1 internalization of wild-type GluA1, but not a mutant GluA1 lacking ubiquitination sites, indicating a crucial role for Eps15 in the trafficking of ubiquitinated AMPARs. These findings reveal novel regulatory mechanisms in the control of glutamate receptor amount and distribution dynamics, which are key factors implicated in higher brain functions and neurological disorders.