With limited efficacy and safety of the methods to treat ischemic stroke (IS), melatonin (МТ) can be considered a promising neuroprotective agent having a pleiotropic mechanism of action. The study aimed to assess the effect of MT contained in original rectal suppositories on the neurological status and microcirculation in the injury focus in experimental acute cerebral ischemia (EACI) in vivo. A total of 30 sexually mature rats were divided into three groups, 10 animals per group: shamoperated (SO) animals; animals with EACI; animals with EACI receiving original rectal suppositories weighing 100 mg with 2.5 mg of melatonin (МТ) throughout 7 days. On days 3 and 7, neurological status was assessed using the Garcia JH score, Placing test, Bederson test; microcirculation rate (MR) was assessed in the brain injury focus by laser flowmetry. A significant decrease in the Garcia JH scores by 58.3% (p = 0.001), Placing Test scores by 57.9% (p = 0.002), along with the significant increase in the Bederson Test scores in animals with EACI compared to SO animals was reported on day 3; the significant decrease in the Garcia JH scores by 75% (p < 0.001), Placing Test scores by 78.9% (p < 0.001) and the significant increase in the Bederson Test scores were reported on day 7. MR decreased by 30% on day 3 (p = 0.02), by 38% on day 7 (p = 0.005). The use of the MT-based rectal suppositories resulted in the neurological deficit restoration in the form of the significant increase in the Gаrcia JH scores by 53.3% (p = 0.008), Placing Test scores by 50% (p = 0.016) and the significant decrease in the Bederson Test scores by 50% (p = 0.029) on day 3;  on day 7, the significant increase in the Garcia JH scores by 233% (p < 0.0001), Placing Test scores by 325% (p < 0.0001) and the significant decrease in the Bederson Test scores by 100% (p < 0.0001) were reported. MR increased by 12.5% on day 3 (p = 0.016), by 43.9% on day 7 (p = 0.005). The correlation analysis revealed the association between the neurological status and MR values: the neurological deficit improvement in animals with EACI in the context of receiving the MT-based rectal suppositories was associated with the MR increase in the ischemic focus in the brain. Thus, partial neurological status restoration in the context of using the MT-based rectal suppositories for EACI resulted from the MT vasoactive properties, which was reflected in the MR increase in the ischemic focus in the brain.