The protective effect of methylene blue (MB) was investigated on the model of focal one-sided traumatic brain injury (TBI) of the sensorimotor cortex region from 1 to 7 days after the injury. TBI caused a reliable disruption of the functions of the limbs contralateral to injury focus, an increase in the expression of S100 protein and blood-brain barrier (BBB) permeability in the ipsilateral hemisphere. The single intravenous injection of MB (1 mg/kg body weight) 30 minutes after TBI significantly reduced the limb function impairment as well as a TBI-induced increase in the expression of inflammatory marker S100 protein, and BBB permeability. When modeling inflammation in vitro, MB was found to protect cultured neurons from the toxic effects of lipopolysaccharide. In conclusion, the preservation of blood-brain barrier and a decrease in the expression of S100 protein may be an important mechanism by means of which MB improves neurological outcome. Our data demonstrate that MB can be a very promising pharmacological compound with neuroprotective properties for TBI treatment.Copyright © 2020. Published by Elsevier B.V.