Aggression is one of the symptoms of methamphetamine (MA) use and withdrawal, which can exacerbate MA addiction and relapse. Many studies have demonstrated that poor sleep is significantly associated with aggression. Melatonin has been indicated to be effective in treating sleep disorders induced by MA, and it can also protect neuronal cells against MA-induced neurotoxicity. However, the underlying effects of melatonin on MA-reduced aggression remain unclarified. This study was designed to evaluate the effects of melatonin on acute MA-induced aggressive behavior in male ICR mice and the effects on neurotransmitters related to aggression. Fifty male ICR mice were randomly assigned to control and treatment groups pretreated with MA (3 mg/kg) or melatonin (2.5, 5, 10 mg/kg) plus MA. Aggressive behaviors were observed through isolation-induced aggression in the resident-intruder model. High-performance liquid chromatography combined with electrochemical detection (HPLC-ECD) was used to anatomize the levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA), and the concentrations of serotonin (5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in the hippocampus involved in behavior processing. The results showed that acute MA administration decreased latency to initial attacks and thereby increased the number and total duration of attacks. Furthermore, HVA level as well as 5-HIAA and 5-HT turnover estimated by 5-HIAA/5-HT ratios declined compared to those in the vehicle group. The medium melatonin pretreatment dose (5 mg/kg) could significantly reverse acute MA-induced aggressive behavior in the form of prolonging latency to initial attacks and thereby attenuating the number of attacks and total duration of attacks. HVA and 5-HIAA levels, 5-HT turnover estimated by 5-HIAA/5-HT ratios, and DA turnover estimated by HVA/DA ratios and (DOPAC + HVA)/DA ratios were elevated compared to those in the MA group. These results indicate that the DA and 5-HT systems are involved in the processes of MA-induced aggressive behaviors and that melatonin has the capacity to reverse MA-induced aggressive behaviors.