In contrast to most secondary active transporters, glutamate uptake into synaptic vesicles is not coupled to an ion but driven instead by membrane potential and regulated allosterically by protons and chloride. To understand the mechanisms, we determined high-resolution criyo-EM structures of VGLUT2 and a mutant in complex with a substrate analogue. We identify previously unrecognized palmitoylation that influences recycling of the transporter. We then used mass spectrometry to identity the lipid modification and the sites modified. We found modification of three N-terminal cysteines (C60, C62 and C64) by palmitic acid.