Breast cancer is the most prevalent cancer among women, posing significant challenges due to its heterogeneity. Recent studies suggest that the ketogenic diet (KD) may enhance chemotherapy efficacy by modulating cancer cell metabolism, particularly through the elevation of ketone bodies like β-hydroxybutyrate (BHB). This study investigates the effects of BHB on breast cancer cells using both 2D and 3D culture models, focusing on its role in developing resistance to fluorouracil (5-FU). We utilized CF41.Mg canine mammary gland cancer cells and MCF7 human breast cancer cells to assess BHB's effects as a pre-treatment and post-treatment under varying glucose conditions. The findings indicated that BHB notably increased cell viability, proliferation, and migration. Pre-treatment resulted in a 52.94 % increase in viability for CF41.Mg cells and a 54.73 % increase for MCF7 cells after 48 h, compared to treatment with 5-FU alone. This enhancement persisted at 72 h, indicating BHB's potential to promote resistance to 5-FU. In 3D spheroid models, which better mimic the tumor microenvironment, BHB pre-treatment significantly increased spheroid size and conferred resistance to 5-FU in both cell lines. Additionally, BHB pre-treatment elevated the expression of proliferation markers such as Ki-67 and tumorigenic markers like MUC-1 (Mucin 1), while showing no significant impact on mesenchymal markers like N-cadherin and vimentin. These findings suggest that BHB significantly increases resistance to 5-FU, indicating that BHB may enable cancer cells to evade chemotherapy-induced stress. Our findings raise important questions about the potential dual role of BHB and KD in promoting cancer cell survival while potentially complicating treatment outcomes.