An imbalance between excitatory (E) glutamate and inhibitory (I) GABA transmission may underlie neurodevelopmental conditions such as Autism Spectrum Disorder (ASD) and schizophrenia. This may be direct, through alterations in synaptic genes, but there is increasing evidence for the importance of indirect modulation of E/I balance through glial mechanisms. Here we used C57BL/6J mice to test the hypothesis that E/I balance can be shifted by N-acetylcysteine (NAC), which acts at the cystine-glutamate antiporter of glial cells. Striatal glutamate was quantified in-vivo using proton magnetic resonance spectroscopy. The effect of NAC on behaviours relevant to ASD was examined in a separate cohort. NAC induced a time-dependent decrease in striatal glutamate, which recapitulated findings of lower striatal glutamate reported in ASD. NAC-treated animals were significantly less active and more ‘anxious’ in the open field test; and NAC-treated females had significantly impaired prepulse inhibition of startle response. This at least partly mimics greater anxiety and impaired sensorimotor gating reported in neurodevelopmental disorders. Thus glial mechanisms regulate E/I balance acutely and have functional consequences. Glial cells may be a potential drug target for the development of new therapies for neurodevelopmental disorders.