d-Serine and glycine are coagonists of NMDA receptors (NMDARs), but their relative contributions for several NMDAR-dependent processes are unclear. We now report that the alanine–serine–cysteine transporter-1 (Asc-1) mediates release of both d-serine and glycine from neurons, and, in turn, this modulates NMDAR synaptic activity. Asc-1 antiporter activity is enhanced by d-isoleucine (d-Ile), which releases d-serine and glycine from Asc-1-transfected cells, primary neuronal cultures, and hippocampal slices. d-Ile has no effect on astrocytes, which do not express Asc-1. We show that d-Ile enhances the long-term potentiation (LTP) in rat and mouse hippocampal CA1 by stimulating Asc-1-mediated endogenous d-serine release. d-Ile effects on synaptic plasticity are abolished by enzymatically depleting d-serine or by using serine racemase knock-out (SR-KO) mice, confirming its specificity and supporting the notion that LTP depends mostly on d-serine release. Conversely, our data also disclose a role of glycine in activating synaptic NMDARs. Although acute enzymatic depletion of d-serine also drastically decreases the isolated NMDAR synaptic potentials, these responses are still enhanced by d-Ile. Furthermore, NMDAR synaptic potentials are preserved in SR-KO mice and are also enhanced by d-Ile, indicating that glycine overlaps with d-serine binding at synaptic NMDARs. Altogether, our results disclose a novel role of Asc-1 in regulating NMDAR-dependent synaptic activity by mediating concurrent non-vesicular release of d-serine and glycine. Our data also highlight an important role of neuron-derived d-serine and glycine, indicating that astrocytic d-serine is not solely responsible for activating synaptic NMDARs.