The glutamate-nitric oxide (NO)-cGMP pathway modulates some forms of learning. How glycine modulates this pathway is unclear. Glycine could modulate the pathway biphasically, enhancing its function through NMDA receptor activation or reducing it through glycine receptor activation. Chronic hyperammonemia impairs the glutamate-NO-cGMP pathway in the cerebellum and induces cognitive impairment. The possible alterations in hyperammonemia of glycinergic neurotransmission and of glutamate-NO-cGMP pathway modulation by glycine remain unknown. The aims were to assess, by in vivo microdialysis in cerebellum: (i) the effects of different glycine concentrations, administered through the microdialysis probe, on the glutamate-NO-cGMP pathway function; (ii) the effects of tonic glycine receptors activation on the pathway function, by blocking them with strychnine; (iii) whether hyperammonemia alters the pathway modulation by glycine; (iv) and whether hyperammonemia alters extracellular glycine concentration and/or glycine receptor membrane expression. In control rats, low glycine levels reduce the pathway function, likely by activating glycine receptors, while 20 μM glycine enhances the pathway function, likely by enhancing NMDA receptor activation. In hyperammonemic rats, glycine did not reduce the pathway function, but enhanced it when administered at 1–20 μM. Hyperammonemia reduces extracellular glycine concentration by approximately 50% and glycine receptor membrane expression. However, tonic glycine receptor activation seems to be enhanced in hyperammonemic rats, as indicated by the larger increase in extracellular cGMP induced by strychnine. These data show that glycine modulates the glutamate-NO-cGMP pathway biphasically and that hyperammonemia strongly alters glycinergic neurotransmission and modulation by glycine of the glutamate-NO-cGMP pathway. These alterations may contribute to the cerebellar aspects of cognitive alterations in hyperammonemia.