Type 2 diabetes can be prevented by lifestyle intervention. We aimed to identify metabolites that associate with glucose metabolism and respond to lifestyle intervention with evidence-based targets for nutrition and physical activity in individuals at high risk of type 2 diabetes. Standard oral glucose tolerance test (OGTT) was used to categorize 624 participants into those having normal glucose tolerance (NGT), isolated impaired glucose tolerance (IGT), IGT with increased fasting glucose (IGT + IFG), and type 2 diabetes. Plasma LC-MS metabolomics was performed to reveal metabolic signatures. The baseline group differences were analysed with the Kruskal–Wallis test and the effect of intervention with a linear mixed-effects model. Significant differences in the metabolite signature were observed between the baseline groups, particularly in amino acids, acylcarnitines, and phospholipids. Fatty acid amides, phospholipids, amino acids, dimethylguanidinovaleric acid, and 5-aminovaleric acid betaine responded most to the lifestyle intervention. Lysophosphatidylcholines containing odd-chain fatty acids showed associations with improved glucose metabolism. Twenty-five metabolites differed between the baseline groups, responded to the intervention, and were associated with changes in glucose metabolism. The findings suggest a metabolite panel could be used in distinguishing individuals with varying degrees of glucose metabolism and in predicting response to lifestyle interventions.