The AMPA receptor (AMPAR) subunits GluR1 and GluR2 show different properties in central neurons and affect AMPAR trafficking via distinct mechanisms. This subunit-specificity is partly achieved by recruiting unique protein modifications on different subunits. Here, we show that palmitoylation of GluR1 and GluR2 subunits also displays subunit-specific properties and functions. Our findings indicate that GluR1 palmitoylation requires dynamic anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus. In contrast, GluR2 subunits are primarily palmitoylated locally in the ER as immature receptors, and an intact microtubule network is required for their palmitoylation. Interestingly, the majority of palmitoylated GluR2 subunits are not associated with GluR1 subunits. We found that preventing palmitoylation results in loss of mature GluR2, but leaves GluR1 intact, as palmitoylation on GluR2 in the ER prevents their sorting to the lysosome after receptor maturation. Moreover, palmitoylation on GluR1 and GluR2 subunits responds differently to neuronal activity. Blocking neuronal activity by tetrodotoxin increased the pool size of palmitoylated GluR2, but not GluR1. Acute stimulation by NMDA and AMPA also differentially affect AMPAR palmitoylation in a subunit-specific manner. The present findings thus indicate that AMPAR palmitoylation is a subunit-specific process that contributes to its regulation and trafficking.