Glutamate (Glu) is the principal excitatory neurotransmitter of the central nervous system. Extracellular Glu increase causes aberrant synaptic signalling leading to neuronal excitotoxicity and death. Glu transmission efficiency depends on the correct functionality and expression of many receptors and transporters, located both on neurons and glial cells. Aberrations in the expression of glutamate receptors, transporters, or metabolizing enzymes were detected in multiple sclerosis (MS) animal models, i.e., experimental autoimmune encephalomyelitis (EAE). In this study, we investigated the effect of olive leaf polyphenols on Glu excitotoxicity. Dark Agouti (DA) rats were used to investigate the effect of olive leaf extract therapy in EAE. Animals were divided into three major groups: control group, EAE-induced group sacrificed on day 20 after EAE induction (EAE), and EAE-induced group treated with OLE therapy (1 g/kg, i.p.) 10 days continuously and sacrificed on the 20th day after induced EAE (EAE+OLE). In addition, the EAE+OLE group consumed olive leaf tea instead of drinking water (1,5% w/v, ad libitum) from the beginning to the end of the experiment. The clinical course was monitored in both groups until the 30th day after EAE induction. The level of glutamate receptors and transporters (GLUR1, NMDAR1, NMDAR2, GLUR5, GRID2, GLAST1, GLT1), antioxidant enzymes (SOD1, SOD2), demyelination/remyelination markers (MBP, TREM2, VAMP2), neurodegeneration (NeuN, SIRT1) and activation markers of microglia (IBA1) were determined by immunoblotting in the lysate of the cerebrum in the rat. Additionally, immunofluorescent staining of GLAST, GLT1, SIRT1 and IBA1 was performed on paraffin-embedded tissues. Our results demonstrated that TOLE attenuated the intensity of EAE-induced symptoms and delays the clinical course of the disease. Furthermore, increased activity of EAAT2/GLT1 glutamate transporter, increased expression of SOD2 antioxidant enzyme, preserved myelin integrity through MBP and TREM2 expression, activativated microglia (IBA1) and preserved neurons (SIRT1) were found in the EAE20+OLE group 20th day after induced EAE in rat brain. This study suggests that TOLE may have a positive impact on the course of treatment of MS.