Vesicular glutamate transporters (VGLUTs) control quantal size of glutamatergic transmission and have been the center of numerous studies over the past two decades. VGLUTs contain two independent transport modes that facilitate glutamate packaging into synaptic vesicles and phosphate (Pi) ion transport into the synaptic terminal. While a transmembrane proton electrical gradient established by a vacuolar-type ATPase powers vesicular glutamate transport, recent studies indicate that binding sites and flux properties for chloride, potassium, and protons within VGLUTs themselves regulate VGLUT activity as well. These intrinsic ionic binding and flux properties of VGLUTs can therefore be modulated by neurophysiological conditions to affect levels of glutamate available for release from synapses. Despite their extraordinary importance, specific and high-affinity pharmacological compounds that interact with these sites and regulate VGLUT function, distinguish between the various modes of transport, and the different isoforms themselves, are lacking. In this review, we provide an overview of the physiologic sites for VGLUT regulation that could modulate glutamate release in an over-active synapse or in a disease state.