Introduction and Objective: Weight management is essential for preventing type 2 diabetes, particularly in individuals with prediabetes. DNA methylation (DNAm) serves as a biomarker for capturing metabolic changes and disease progression. This study aimed to discover DNAm biomarkers linked to weight loss response in diabetes prevention interventions for individuals with prediabetes. Methods: We analyzed 168 subjects (ages 30 to 70, BMI ≥ 23 kg/m2) from the KDPS cohort, all with prediabetic conditions. Participants underwent a 6-month intervention in three groups: standard management (STM), lifestyle modification (LSM), metformin (MET). Blood DNAm was assessed using Illumina EPIC v2.0 arrays (697,649 CpG sites post-QC). Differential methylation loci were identified between the 3% weight-loss responders and those who maintained weight post-intervention using SeSAMe package. Results: Weight loss was significantly higher in LSM (1.8 times, p=0.052), and MET (2 times, p=0.028), compared to STM. Among the 3% weight-loss responders, 220 hypermethylated and 1,912 hypomethylated CpG sites exhibited specific DNAm changes (Δβ ≤ 0.03, FDR < 0.05). Reactome pathway analysis highlighted Notch signaling pathway, associated with T2DM and obesity, involving HDAC4, NOTCH2, and NCOR2 genes. Four CpG sites (cg03404471 [OXCT1], cg27133681 [CAMK2G], cg13006177 [SAMD12], cg23968558 [RAI1]) displayed differential methylation between weight-loss responders and weight maintainers (Effect size ≤ 0.02, FDR < 0.1). Conclusion: These findings highlight specific DNAm changes associated with weight loss in diabetes prevention. Long-term studies are needed to validate these findings and explore their potential in personalized diabetes prevention strategies. Disclosure S. Song: None. G. Kim: None. S. Chon: None. A.M. Lindroth: None. J. Lim: None. D. Yoon: None. Y. Park: None. Funding Korea National Institute of Health (2023-NI-006-01)