In experimental autoimmune encephalomyelitis (EAE), a large number of myeloid-derived suppressor cells (MDSCs) appear, and these cells can accelerate the development of EAE in mice. The aim of this study was to evaluate pseudolycorine chloride’s (PLY) inhibitory effect on the expansion of MDSCs and differentiation of monocyte-like MDSCs (M-MDSCs), while proving that PLY can improve the severity of Th17 cell-mediated EAE by affecting MDSCs. Flow cytometry suggested that PLY significantly inhibited the proliferation and expansion of MDSCs stimulated by IL-6 and GM-CSF, especially the differentiation of M-MDSCs in a dose-dependent manner. Three concentrations of PLY (0.67, 2, and 6 μM) had no cytotoxicity in vitro . Hematoxylin and eosin (H&E) staining, LFB, and immunofluorescence suggested inflammatory infiltration and demyelination. MDSCs and related Th17 cell infiltration in the spinal cord were inhibited by PLY (40 mg/kg). In conclusion, PLY inhibited the differentiation and expansion of MDSCs, especially M-MDSCs, and intervened in the progression of EAE in mice.