Inadequate pancreatic beta cell function underlies type 1 and type 2 diabetes mellitus. Strategies to expand functional cells have focused on discovering and controlling mechanisms that limit the proliferation of human beta cells. Here, we developed an engraftment strategy to examine age-associated human islet cell replication competence and reveal mechanisms underlying age-dependent decline of beta cell proliferation in human islets. We found that exendin-4 (Ex-4), an agonist of the glucagon-like peptide 1 receptor (GLP-1R), stimulates human beta cell proliferation in juvenile but not adult islets. This age-dependent responsiveness does not reflect loss of GLP-1R signaling in adult islets, since Ex-4 treatment stimulated insulin secretion by both juvenile and adult human beta cells. We show that the mitogenic effect of Ex-4 requires calcineurin/nuclear factor of activated T cells (NFAT) signaling. In juvenile islets, Ex-4 induced expression of calcineurin/NFAT signaling components as well as target genes for proliferation-promoting factors, including NFATC1, FOXM1, and CCNA1. By contrast, expression of these factors in adult islet beta cells was not affected by Ex-4 exposure. These studies reveal age-dependent signaling mechanisms regulating human beta cell proliferation, and identify elements that could be adapted for therapeutic expansion of human beta cells.