The study aims to explore the molecular mechanisms of 5-Fluorouracil in the development of diabetes, specifically focusing on DNA hypermethylation. Previous research has suggested that 5-FU, a chemotherapeutic drug, induces hyperglycemia in patients. A network pharmacology-based approach was used to identify target proteins for 5-FU using STITCH, ChEMBL and Cytoscape databases. Docking studies were conducted on 5- FU against target proteins. In-vivo studies were performed on Swiss albino mice, where the treatment group received 5-FU (20 mg/kg, intraperitoneally). Fasting blood sugar levels were measured at intervals of 4, 8, 12, 16, and 20 days. According to the network pharmacology result TXNIP, EGFR and DNMT1 were identified as validated targets for 5-FU in hypermethylation and hyperglycemia. In-silico docking studies revealed that 5-FU displayed a binding energy of -4.7 kcal/mol, -4.9 kcal/mol, and -5.6 kcal/mol against target proteins TXNIP, EGFR and DNMT1 respectively. In in vivo studies the treatment group showed a significant increase in fasting blood sugar levels compared to the normal control group. Where n=6, ***p<0.001. Histopathological analysis confirmed the pancreatic degenerative response induced by 5-FU. The study suggests that 5-FU induces DNA hypermethylation, impacting glucose regulation through proteins like TXNIP, EGFR and DNMT1.