Objective: This study aims to investigate the effects of Vitamin D (Vit D) on DNA methylation in Alzheimer's Disease (AD) cell models, and the relationship between these effects and AD pathological progression, to provide new insights and potential targets for AD prevention and treatment. Methods: An AD cell model was constructed by treating human neuroblastoma SH-SY5Y cells with Aβ1-42 (10 μmol·L-1) for 24 hours. Western blot was used to detect p-tau protein expression to validate the model. Cells were divided into normal control group, AD model group, and high, medium, and low concentration Vit D treatment groups. MTT assay was used to evaluate the effect of Vit D on cell proliferation. Real-time PCR and Western blot were used to detect mRNA and protein expression levels of DNA methyltransferases, respectively. Results: The AD model group showed significantly increased p-tau protein expression. Vit D treatment groups demonstrated significantly improved cell proliferation rates compared to the AD model group, in a dose-dependent manner. Compared with the AD model group, the mRNA and protein expression levels of DNMT1 and DNMT3A were significantly upregulated in the Vit D treatment groups. Conclusion: This study reveals that Vit D improves pathological changes in AD cell models by upregulating DNMT1 and DNMT3A expression, thereby increasing DNA methylation levels. This finding provides a new perspective for understanding the protective mechanism of Vit D in AD, while also offering potential epigenetic targets for the development of AD prevention and treatment strategies.