Objectives <p>Extracellular vesicles (EVs) are implicated in the pathophysiology of obesity and diabetes. This study aimed to analyze the impact of diabetes on EV protein dynamics in patients with obesity before and after bariatric surgery.</p> Methods <p>This prospective study assessed the serum EV proteins in 30 patients with obesity, with and without diabetes, and 37 healthy controls using liquid chromatography-tandem mass spectrometry. EV proteins were also analyzed 6 months after bariatric surgery in patients with obesity.</p> Results <p>We found 19 differentially expressed proteins (DEPs) in EVs between healthy controls and patients with obesity. In addition, 20 DEPs between patients with obesity who had and did not have diabetes were identified. After bariatric surgery, 14 DEPs mainly involved in the immune system were identified in patients with obesity and diabetes; adiponectin (ADIPOQ), mannose binding lectin 2 (MBL2), and hornerin (HRNR) were associated with glycemic control, body mass index, and weight loss, respectively. In patients with obesity without diabetes, 13 DEPs after bariatric surgery were predominantly involved in reactive oxygen species metabolism.</p> Conclusions <p>Diabetes affects the differences in EV protein profiles in patients with obesity before and after bariatric surgery. These DEPs may serve as potential biomarkers and therapeutic targets for obesity-related diabetes.</p>