Abstract: Objective To analyze the serum metabolomic characteristics of patients with early-onset type 2 diabetes (T2DM) and their interactions with gut microbiota. Methods A total of 363 early-onset T2DM patients treated at Luoyang Central Hospital Affiliated to Zhengzhou University from July 2021 to September 2024 were selected, and 300 healthy volunteers who underwent physical examinations during the same period were chosen as a control group. Clinical data were collected for analysis. The early-onset T2DM patients were divided into three groups: overweight early-onset T2DM (group A, n=150), obese early-onset T2DM (group B, n=117), and normal BMI early-onset T2DM (group C, n=96). In each group (A, B, C, and healthy control group D), 25, 24, 22, and 25 patients were randomly selected for LC-MS untargeted metabolomics analysis and 16S rRNA sequencing. The serum metabolomic characteristics of each group and their interactions with gut microbiota were analyzed. Results Significant differences were observed in clinical indicators, such as sex, subcutaneous fat area, visceral fat area, waist circumference (WC), hip circumference (HC), waist-to-hip ratio (WHR), uric acid (UA), 2-hour postprandial blood glucose (2hPG), fasting C-peptide (FCP), 120-minute C-peptide (C-P120), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) among the early-onset T2DM subgroups (P<0.05). LC-MS untargeted metabolomics and 16S rRNA sequencing results revealed that groups A, B, and C shared 436 differential metabolites and 5 differential microbiota with group D, and these microbiota and metabolites were correlated. For instance, Faecalibacterium was significantly positively correlated with the metabolite new andrographolide, while Ruminococcus was negatively correlated with the pro-inflammatory lipid palmitoylglucuronic acid. Moreover, the differential metabolites were also correlated with clinical indicators of early-onset T2DM. For example, C-P120 was negatively correlated with MGTS(18:0/0:0); fasting blood glucose (FPG) was positively correlated with DL-2-hydroxy stearic acid; and 2hPG was negatively correlated with 5α-androstan-17β-ol-3-one glucuronide. Conclusions Early-onset T2DM patients exhibit 436 serum differential metabolites and five characteristic gut microbiota imbalances. These microbiota-metabolite interactions are significantly associated with glucose and lipid metabolic disorders, providing new biomarkers and potential intervention targets for early disease diagnosis.