Methamphetamine (MA) is a psychostimulant and addictive substance. Long-term uses and toxic high doses of MA can induce neurotoxicity. The present study aimed to investigate the protective role of melatonin against MA toxicity-induced dysregulation of the neurotransmission related to cognitive function in rats. The adult male Sprague Dawley rats were intraperitoneally injected with 5 mg/kg MA for 7 consecutive days with or without subcutaneously injected with 10 mg/kg melatonin before MA injection. Some rats were injected with saline solution (control) or 10 mg/kg melatonin. MA administration induced reduction in total weight gain, neurotoxic features of stereotyped behaviors, deficits in cognitive flexibility, and significantly increased lipid peroxidation in the brain which diminished in melatonin pretreatment. The neurotoxic effect of MA on glutamate, dopamine and GABA transmitters was represented by the alteration of the GluR1, DARPP-32 and parvalbumin (PV) levels, respectively. A significant decrease in the GluR1 was observed in the prefrontal cortex of MA administration in rats. MA administration significantly increased the DARPP-32 but decreased PV in the striatum. Pretreatment of melatonin can abolish the neurotoxic effect of MA on neurotransmission dysregulation. These findings might reveal the antioxidative role of melatonin to restore neurotransmission dysregulation related to cognitive deficits in MA-induced neurotoxicity. © 2021