Most cases of hyperinsulinism of infancy (HI) are caused by mutations in either the sulfonylurea receptor-1 (SUR1) or the inward rectifying K⁺ channel Kir6.2, two subunits of the β-cell ATP-sensitive K⁺ channel (K_ATP channel). Histologically, HI can be divided into two major subtypes. The diffuse form is recessively inherited and involves all β-cells within the pancreas. Focal HI consists of adenomatous hyperplasia within a limited region of the pancreas, and it is caused by somatic loss of heterozygosity (LOH), including maternal Ch11p15-ter in a β-cell precursor carrying a germ-line mutation in the paternal allele of SUR1 or Kir6.2. Several imprinted genes are located within this chromosomal region, some of which, including p57^KIP2 and IGF-II, have been associated with the regulation of cell proliferation. Using double immunostaining, we examined p57^KIP2 expression in different islet cell types, in control pancreases from different developmental stages (n = 15), and in pancreases from patients with both diffuse (n = 4) and focal HI (n = 9). Using immunofluorescence and computerized image analysis, we quantified IGF-II expression in β-cells from patients with focal HI (n = 8). Within the pancreas, p57^KIP2 was specifically localized to the endocrine portion. β-Cells demonstrated the highest frequency of expression (34.9 ± 2.7%) compared with ∼1–3% in other cell types. The fraction of β-cells expressing p57^KIP2 did not vary significantly during development. β-Cells within the focal lesions did not express p57^KIP2, whereas IGF-II staining inside focal lesions was mildly increased compared with unaffected surrounding tissue. In conclusion, we demonstrate that p57^KIP2 is expressed and is paternally imprinted in human pancreatic β-cells. Loss of expression in focal HI is caused by LOH and is associated with increased proliferation and increased IGF-II expression. Manipulation of p57^KIP2 expression in β-cells may provide a mechanism by which proliferation can be modulated, and thus this gene is a potential therapeutic target for reversing the β-cell failure observed in diabetes.