Three isoforms of a vesicular glutamate transporter (VGLUT1–3) have been identified. Of these, VGLUT1 is the major isoform in the cerebral cortex and hippocampus where it is selectively located on synaptic vesicles of excitatory glutamatergic terminals. Variations in VGLUT1 expression levels have a major impact on the efficacy of glutamate synaptic transmission. Given evidence linking alterations in glutamate neurotransmission to various neuropsychiatric disorders, we investigated the possible influence of a down‐regulation of VGLUT1 transporter on anxiety, depressive‐like behaviour and learning. The behavioural phenotype of VGLUT1‐heterozygous mice (C57BL/6) was compared to wild‐type (WT) littermates. Moreover, VGLUT1–3 expression, hippocampal excitatory terminal ultrastructure and neurochemical phenotype were analysed. VGLUT1‐heterozygous mice displayed normal spontaneous locomotor activity, increased anxiety in the light–dark exploration test and depressive‐like behaviour in the forced swimming test: no differences were shown in the elevated plus‐maze model of anxiety. In the novel object recognition test, VGLUT1^+/– mice showed normal short‐term but impaired long‐term memory. Spatial memory in the Morris water maze was unaffected. Western blot analysis confirmed that VGLUT1 heterozygotes expressed half the amount of transporter compared to WT. In addition, a reduction in the reserve pool of synaptic vesicles of hippocampal excitatory terminals and a 35–45% reduction in GABA in the frontal cortex and the hippocampus were observed in the mutant mice. These observations suggest that a VGLUT1‐mediated presynaptic alteration of the glutamatergic synapses, in specific brain regions, leads to a behavioural phenotype resembling certain aspects of psychiatric and cognitive disorders.