Context:Arginine, its methylated metabolites, and other metabolites related to the urea cycle have been independently associated with cardiovascular risk, but the potential causal meaning of these associations (positive for some metabolites and negative for others) remains elusive due to a lack of studies measuring metabolite changes over time. Objective:To examine the association between baseline and 1-year concentrations of urea cycle metabolites and cardiovascular disease (CVD) in a case-cohort setting. Design:A case-cohort study was nested within the Prevención con Dieta Mediterránea trial. We used liquid chromatography-tandem mass spectrometry to assess metabolite levels at baseline and after 1-year follow-up. The primary CVD outcome was a composite of myocardial infarction, stroke and cardiovascular death. We used weighted Cox regression models (Barlow weights) to estimate multivariable-adjusted hazard ratios (HRs) and their 95% confidence intervals (CIs). Setting:Multicenter randomized trial in Spain. Participants:Participants were 984 participants accruing 231 events over 4.7 years' median follow-up. Main Outcome Measure:Incident CVD. Results:Baseline arginine/asymmetric dimethylarginine ratio [HR per standard deviation (SD) = 0.80; 95% CI, 0.67 to 0.96] and global arginine availability [arginine / (ornithine + citrulline)] (HR per SD = 0.83; 95% CI, 0.69 to 1.00) were significantly associated with lower risk of CVD. We observed no significant association for 1-year changes in these ratios or any effect modification by the Mediterranean diet (MD) intervention. Conclusions:A higher baseline arginine/asymmetric dimethylarginine ratio was associated with lower CVD incidence in a high cardiovascular risk population. The intervention with the MD did not change 1-year levels of these metabolites.