To investigate effect of vitamin D receptor (VDR) promoter DNA methylation on the course of experimental autoimmune encephalomyelitis (EAE). Methods: C57BL/6 female mice were selected to the control group (n=12) or EAE group (n=48). The EAE model was established in the EAE group by injection of MOG35-55. The control group received no treatment. Using the 15-point Weaver’s neurological function scoring method, the EAE group was divided into the 0 score (n=10), high score (n=10), and low score (n=10) subgroups. Quantitative real time PCR (qPCR) and Western blot (WB) were used to detect the expression level of VDR in the lumbar spinal cord of mice in each group. Bisulfite genomic sequencing (BSP) was used to detect the DNA methylation level of the VDR promoter in each group. Results: The myelin sheath structure of the 0 score, high score, and low score subgroup mice was observed under transmission electron microscope and showed different degrees of damage when compared to the control group. The myelin sheath ring structure of the 0 score and low score subgroups was intact, and that of the high score subgroup disappeared. In the spinal cord, the expression of VDR protein in the EAE 0 score, high score, and low score subgroups was lower than that in the control group (P0=0.0046, Phigh=0.0088, Plow<0.0001); in the low score subgroup was further lower than in the 0 score subgroup (P=0.0032), and in the low score subgroup was lower than in the high score subgroup (P= 0.0134). The expression of VDR mRNA in each EAE subgroup also showed a downward trend compared to that in the control group (P0>0.05, Phigh=0.0439, Plow=0.0349); compared with expression in the 0 score subgroup, that in the low score subgroup decreased significantly (P=0.0142). The DNA methylation level of the VDR promoter in each group was low, there was no change in trend, and the difference was not statistically significant (P>0.05). Conclusion: VDR was expressed differently in different neural function score groups of the EAE model, and the methylation of VDR promoter DNA was not the main reason for these differences.