Widespread application of β-cell replacement strategies for diabetes is dependent upon the availability of an unlimited supply of cells exhibiting appropriate glucose-responsive insulin secretion. Therefore, a great deal of effort has been focused on understanding the factors that control β-cell growth. Previously, we found that human β-cell-enriched islet cultures can be stimulated to proliferate, but expansion was limited by growth arrest after 10-15 cell divisions. Here, we have investigated the mechanism behind the growth arrest. Our studies, including analyses of the expression of senescence-associated β-galactosidase, p16(INK)4a levels, and telomere lengths, indicate that cellular senescence is responsible for limiting the number of cell divisions that human β-cells can undergo. The senescent phenotype was not prevented by retrovital transduction of the hTERT gene, although telomerase activity was induced. These results have implications for the use of primary human islet cells in cell transplantation therapies for diabetes.