Cocaine seeking behaviour and relapse have been linked to impaired potentiation and depression at excitatory synapses in the nucleus accumbens, but the mechanism underlying this process is poorly understood. We show that, in the rat nucleus accumbens core, D-serine is the endogenous coagonist of N-methyl-D-aspartate receptors, and its presence is essential for N-methyl-D-aspartate receptor-dependent potentiation and depression of synaptic transmission. Nucleus accumbens core slices obtained from cocaine-treated rats after 1 day of abstinence presented significantly reduced D-serine concentrations, increased expression of the D-serine degrading enzyme, D-amino acid oxidase, and downregulated expression of serine racemase, the enzyme responsible for D-serine synthesis. The D-serine deficit was associated with impairment of potentiation and depression of glutamatergic synaptic transmission, which was restored by slice perfusion with exogenous D-serine. Furthermore, in vivo administration of D-serine directly into the nucleus accumbens core blocked behavioural sensitization to cocaine. These results provide evidence for a critical role of D-serine signalling in synaptic plasticity relevant to cocaine addiction.