This study aims to elucidate the molecular mechanisms linking dietary stress from a high-fat simple carbohydrate (HFSC) diet to an elevated risk of pancreatic cancer. Using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS), we performed a comprehensive proteomic analysis of collagenase-digested pancreatic islets from HFSC-fed mice. The study identified a significant upregulation and downregulation of 33 and 34 proteins respectively in HFSC group compared to the control group. The results revealed substantial alterations in the expression of proteins involved in key biological processes such as metabolic stress, protein synthesis, and endoplasmic reticulum (ER) stress, all of which are crucial for maintaining cellular homeostasis under dietary stress. Notably, the study identified several potential biomarkers, including MANF, VCP, HYOU1, and PRDX1, which are intricately linked to pancreatic function and the progression of cancer. These biomarkers play pivotal roles in managing cellular stress responses, protein folding, and metabolic regulation, offering insights into how a HFSC diet contributes to pancreatic carcinogenesis. This research highlights the significant impact of dietary factors on pancreatic cancer development and underscores the potential of these biomarkers as therapeutic targets for early detection and treatment, ultimately aiming to improve patient outcomes in PDAC.