The islets of Langerhans, micro-organs for maintaining glucose homeostasis, range in size from small clusters of <10 cells to large islets consisting of several thousand endocrine cells. Islet size distributions among various species are similar and independent of body size, suggesting an intrinsic limit to islet size. Little is known about the mechanisms regulating islet size. We have carried out a comprehensive analysis of changes of islet size distribution in the intact mouse pancreas from birth to eight months, including mathematical modeling to quantify this dynamic biological process. Islet growth was size-dependent during development, with preferential expansion of smaller islets and fission of large interconnected islet-like structures occurring most actively at approximately three weeks of age at the time of weaning. The process of islet formation was complete by four weeks with little or no new islet formation thereafter, and all the β-cells had low proliferation potential in the adult, regardless of islet size. Similarly, in insulinoma-bearing mice, the early postnatal developmental process including fission followed the same time course with no new islet formation in adults. However, tumor progression led to uncontrolled islet growth with accelerated expansion of larger islets. Thus, islet formation and growth is a tightly regulated process involving preferential expansion of small islets and fission of large interconnected islet-like structures.