The metabolic response to dietary fat intake and its association with prediabetes are unclear. We aimed to identify the plasma metabolite profiles related to dietary fats and test if they are associated with prediabetes risk. Within the Precision Nutrition and Food Safety for Dietary Prevention of Chronic Disease cohort, we analyzed plasma metabolites from 207 participants (comprising 102 prediabetes cases and 105 controls) via untargeted metabolomics. Dietary fat intakes were assessed using a validated food frequency questionnaire. Metabolic signatures related to dietary fats were identified using elastic net regression and 10-fold cross-validation. The associations of metabolic signatures with prediabetes were evaluated using Poisson regression. We identified multi-metabolite profiles comprising 26 metabolites for intake of saturated fatty acids (SFAs), 32 metabolites for monounsaturated fatty acids (MUFAs), 27 metabolites for polyunsaturated fatty acids (PUFAs), 24 metabolites for n-3 PUFAs, and 25 metabolites for n-6 PUFAs. These metabolic signatures were robustly correlated with corresponding dietary fats intakes (Pearson r = 0.42–0.61; P < 0.001). After adjusting for potential risk factors, metabolic signatures of PUFAs (OR_{Q4 vs. Q1} = 0.57, 95% CI: 0.35–0.93) and n-3 PUFAs (OR_{Q4 vs. Q1} = 0.56, 95% CI: 0.34–0.92) were inversely associated with prediabetes prevalence but no significant associations were found for metabolic signatures of dietary SFAs, MUFAs, and n-6 PUFAs. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that dietary PUFAs and n-3 PUFAs promoted the pentose phosphate pathway and glycerolipid metabolism. Moreover, key metabolites related to PUFA/n-3 PUFA consumption were correlated with prediabetes. Our findings reveal the plasma metabolic signatures characterizing typical dietary fat intake and support the beneficial role of PUFAs, especially n-3 PUFAs, in prediabetes prevention by modulating the pentose phosphate pathway and glycerolipid metabolism.