Experimental autoimmune encephalomyelitis (EAE) is a laboratory model for multiple sclerosis (MS), an autoimmune disease characterized by chronic inflammation in the CNS, associated with demyelination and neurological disorders. Dysbiosis of the gut microbiota is a risk factor for MS. Probiotic strains can modulate the microbiota composition and are considered a therapeutic approach for MS.This study aimed to explore the therapeutic potential of Enterococcus faecium L-3 (E.faecium), a probiotic strain, in modulating gut microbiota, intestinal morphology, and immune responses within an EAE rat model.The study was conducted on female Wistar rats. EAE was induced by injection of an encephalitogenic mixture. From days 2 to 16 post-induction, the rats received saline (control) or E.faecium orogastrically. Disease severity was assessed through clinical indices. Gut microbiota composition was studied by PCR and sequencing, intestinal microstructure via electron microscopy, immune cell profiling by flow cytometry, and cytokine levels through ELISA and RT-qPCR. p < 0.05.Rats receiving E.faecium showed reduced disease duration and severity, accompanied by significant microbiota shifts at the phylum level during EAE's peak, including increased Bacteroidota in treated rats and decreased Bacillota (Firmicutes), without affecting other phyla. E.faecium treated rats exhibited better intestinal epithelium recovery with fewer damaged microvilli areas compared to the control. Investigation into immune cell phenotypes and cytokine levels revealed modifications in circulating immune cells, with an increase in B-cells and dynamic changes in T-cell subsets, as well as increased anti-inflammatory response in E.faecium treated rats, indicating a potential immune modulation mechanism behind the observed EAE attenuation.