We investigated the cut-off point of the Matsuda Index in Japanese according to the guideline from the Clinical and Laboratory Standards Institute. A total of 1,596 subjects free from medications for diabetes mellitus, dyslipidemia and/or hypertension, and without cardiovascular diseases or chronic renal failure underwent a health check-up and oral glucose tolerance test (OGTT). We recruited 204 healthy reference individuals with normal glucose tolerance without obesity, any component of metabolic syndrome or elevated alanine aminotransferase. The Matsuda Index was calculated with 0- and 120-min data during OGTT. As the index was not normally distributed (P < 0.001 by the Shapiro–Wilk test), the log-transformed value (P = 0.876 by the Shapiro–Wilk test) was used. The mean ± 2 standard deviations were taken as the reference limits. The lower reference limit of the Matsuda Index was then calculated to be 4.3. Our result shows that a Matsuda Index <4.3 indicates the presence of insulin resistance in Japanese.