Type 2 diabetes (T2D) is characterized by a steady worsening of β-cell dysfunction as the disease progresses. The objective of this study was to estimate the decline of insulin secretion in Japanese type 2 diabetic patients (T2D-patients) by glucagon injection over an observation period of more than 10 years. Thirty-three T2D-patients were followed for 10.4 ± 1.4 years. Fasting C-peptide immunoreactivity (FCPR), the 6 min value of CPR after glucagon injection (6MCPR), and the increment of CPR (ΔCPR) were measured at baseline and follow-up. FCPR, 6MCPR, ΔCPR were significantly lower at follow-up than at baseline (p<0.05, p<0.005, and p<0.0005, respectively). The annual change of ΔCPR was significantly (p<0.05) greater than the annual change of FCPR (-0.062 ± 0.076 ng/mL/year and -0.025 ± 0.067 ng/mL/year, respectively). In contrast, CPR-index (an index of β-cell function) and SUIT-index (secretory units of islets in transplantation) calculated based on fasting blood samples were unaltered. The annual changes of FCPR, 6MCPR, and ΔCPR were negatively correlated with the FCPR, 6MCPR, and ΔCPR values at baseline, respectively. Duration of diabetes, BMI, diabetic retinopathy, and secondary sulfonylurea failure at baseline were not correlated with the annual changes of FCPR, 6MCPR, and ΔCPR. In conclusion, our longitudinal observations suggest that β-cell function progressively declines in Japanese T2D-patients. The annual declines of ΔCPR were more prominent than the annual declines of FCPR. ΔCPR after glucagon injection may be more useful for estimating individual longitudinal insulin secretion than fasting blood samples.