Abstract Breast Cancer (BC) is a widespread malignancy that affects the lives of millions of women each year. Prevention of BC through administration of epigenetically active dietary phytochemicals has received increased interest in recent years due to its relatively low cost, few side effects and potential to reduce the financial and healthcare burdens associated with BC. Many phytochemicals’ anticancer functions are thought to arise from their effects on the epigenome, although the relative importance of specific genes’ epigenetic states remain unclear. To elucidate this, we utilized human BC cell lines in combination with CRISPR-dCas technology tied to methylation modifiers to explore the importance of gene specific DNA methylation to cancer cell phenotype in both naïve BC cells and BC cells treated with the phytochemical Withaferin A (WA). We found that demethylation of promoters of the tumor suppressors p21 and p53 results in increased gene expression, while methylation of the promoter of the oncogene CCND1 results in decreased in gene expression. Additionally, changes to biological processes were associated with tumor suppressor promoter methylation state, including increases in cell cycle arrest for a demethylated p21 promoter and decreases in cell viability for a demethylated p53 promoter. We also found that downregulation of p21 through targeting DNMT3A to its promoter was able to ablate the anticancer function of WA in both MDA-MB-231 and MCF-7 cells. Taken together, these results highlight the potential importance of DNA methylation changes for cancer cell phenotype and a possible mechanism of action for the phytochemical WA in BC prevention. Citation Format: Andrew Brane, Trygve O. Tollefsbol. The importance of DNA methylation to breast cancer cell phenotype and its role in the anticancer activity of withaferin-A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6022.