The mass of pancreatic β-cells is maintained throughout lifetime to control blood glucose levels. Although the major mechanism of the maintenance of β-cell mass after birth is thought to be selfreplication of pre-existing β-cells, it is possible that pancreatic β-cells are also generated from non-β-cells. Here, we address this issue by using the inducible Cre/loxP system to trace β-cells. We generated Ins2-CreERT2/R26R-YFP double knock-in mice, in which pancreatic β-cells can be labeled specifically and permanently upon injection of the synthetic estrogen analog tamoxifien, and then traced the β-cells by pulse and chase experiment in several different conditions. When β-cells were labeled in adults under physiological and untreated conditions, the frequency of the labeling (labeling index) was not altered significantly throughout the 12-month experimental period. In addition, the labeling index was not changed after ablation of β-cells by streptozotocin treatment. However, when tamoxifen was injected to pregnant mothers just before they gave birth, the labeling index in the neonates was decreased significantly around weaning, suggesting that β-cells are generated from non-β-cells. These results indicate that various mechanisms are involved in the maintenance of β-cells after birth, and that the present system using knock-in mice is useful for investigation of β-cell fate.