Glutamate is the major excitatory transmitter in the vertebrate nervous system. To maintain synaptic efficacy, recycling synaptic vesicles (SV) are refilled with glutamate by vesicular glutamate transporters (VGLUTs). The dynamics and mechanism of glutamate uptake in intact neurons are still largely unknown. Here, we show by live-cell imaging with pH- and chloride-sensitive fluorescent probes in cultured hippocampal neurons of wild-type and VGLUT1-deficient mice that in SVs VGLUT functions as a glutamate/proton exchanger associated with a channel-like chloride conductance. After endocytosis most internalized Cl- is substituted by glutamate in an electrically, and presumably osmotically, neutral manner, and this process is driven by both the Cl- gradient itself and the proton motive force provided by the vacuolar H+-ATPase. Our results shed light on the transport mechanism of VGLUT under physiological conditions and provide a framework for how modulation of glutamate transport via Cl- and pH can change synaptic strength.