Amino acids have emerged as novel biomarkers for predicting type 2 diabetes (T2D), but the epidemiologic data linking circulating amino acid profiles with T2D are sparse in Asian populations. We conducted a nested case-control study within a cohort of 4,754 nondiabetic Japanese employees who attended a comprehensive health checkup in 2008-2009 and agreed to provide blood samples. During a 5-year follow-up, incident T2D cases were ascertained based on plasma glucose, glycated hemoglobin, and self-report. Two controls matched to each case on sex, age, and the date of serum sampling were randomly selected by using density sampling, resulting in 284 cases and 560 controls with amino acid measures. High concentrations of valine, leucine, isoleucine, phenylalanine, tyrosine, alanine, glutamate, ornithine, and lysine were associated with an increased risk of incident T2D, in a linear manner. High glutamine concentrations were associated with a decreased risk of incident T2D. Further adjustment for the homeostasis model assessment of insulin resistance attenuated these associations. Overall, these amino acids may be novel useful biomarkers in the identification of people at risk of T2D before overt symptoms. Insulin resistance may account for or mediate the relationship between these amino acids and risk of incident T2D.