We aimed to examine the association between impaired proinsulin processing in pancreatic beta cells and type 2 diabetes mellitus in non-obese Japanese patients. Participants were divided into groups for normal glucose tolerance, prediabetes, and type 2 diabetes based on the oral glucose tolerance test (OGTT). Activities of prohormone convertase (PC) 1/3 and PC2 in fasting states were estimated. Multiple regression analysis was undertaken to ascertain if alteration of the activities of these enzymes contributes to the development of impaired glucose tolerance by comparison with HOMA-β and the oral disposition index (DI(O)). Overall, 452 subjects were included. PC1/3 activity tended to decrease in type 2 diabetes compared with normal glucose tolerance. PC2 activity showed no difference among the three groups. Decreased estimated PC1/3 activity was significantly associated with type 2 diabetes after adjustment for sex, age, creatinine, triglycerides, HOMA-β and DI(O). Odds ratios (95% CI) of PC1/3, HOMA-β, and DI(O) were 2.16 (1.12-4.19), 3.44 (1.82-6.52) and 14.60 (7.87-27.11), respectively. Furthermore, decreased PC1/3(≤1.7) combined with decreased HOMA-β (≤30) had a sensitivity of 73% and specificity of 62%. Decreased PC1/3 activity may be a useful measurement of beta-cell function alongside decreased HOMA-β or DI(O). A combined decrease in estimated fasting PC1/3 activity and HOMA-β measurement led to suspicion of type 2 diabetes in the non-obese Japanese population studied.