Several amino acids (AAs) have been shown to be associated with insulin resistance and increased risk of type 2 diabetes, but no previous studies have investigated the association of AAs with insulin secretion in a longitudinal setting. Our study included 5,181 participants of the cross-sectional METabolic Syndrome In Men (METSIM) study having metabolomics data on 20 AAs. A total of 4,851 had a 7.4-year follow-up visit. Nine AAs (phenylalanine, tryptophan, tyrosine, alanine, isoleucine, leucine, valine, aspartate, and glutamate) were significantly ( < 5.8 × 10) associated with decreases in insulin secretion (disposition index) and the elevation of fasting or 2-h glucose levels. Five of these AAs (tyrosine, alanine, isoleucine, aspartate, and glutamate) were also found to be significantly associated with an increased risk of incident type 2 diabetes after adjustment for confounding factors. Our study is the first population-based large cohort to report that AAs are associated not only with insulin resistance but also with decreased insulin secretion.