The glucokinase-induced up-regulation of insulin receptor substrate 2 (IRS-2) plays an important role in β-cell adaptive proliferation in response to high-fat diet-induced insulin resistance. This study aimed to investigate the role of IRS-2 in the proliferation of β-cells after a 60% partial pancreatectomy. IRS-2-deficient (IRS-2(-/-)) mice or wild-type mice were subjected to a pancreatectomy (60% partial pancreatectomy) or a sham operation (Sham). The β-cell proliferation and gene expression profiles of the islets were then assessed. Gene expression in islets from pancreatectomized and Sham C57BL/6J male mice was analyzed using a cDNA microarray analysis. To compare with β-cell proliferation induced by a high-fat diet, Gck(+/-) mice subjected to a pancreatectomy were also analyzed. The IRS-2(-/-) mice exhibited β-cell expansion and a significant increase in β-cell proliferation after the pancreatectomy, compared with the Sham group. Although glucose-stimulated insulin secretion from islets was not impaired, IRS-2(-/-) mice manifested severe hyperglycemia after the pancreatectomy. The expression levels of Aurora kinase B, Cyclin A, and Cyclin B1 in the pancreatectomized islets were also enhanced in the IRS-2(-/-) mice. A gene set enrichment analysis suggested an association between the genes that were up-regulated in the pancreatectomized islets and those involved in M phase progression in the cell cycle. β-Cell proliferation after a pancreatectomy was observed even in the Gck(+/-) mice. In conclusion, IRS-2 was not required for β-cell proliferation but might be needed for functional β-cell mass, after a pancreatectomy. A partial pancreatectomy in mice may be an attractive model for the development of new strategy for exploring the unique nature of β-cell proliferation.