Plasminogen activator inhibitor–1 (PAI-1) has been shown to be a key component of the senescence-related secretome and a direct mediator of cellular senescence. In murine models of accelerated aging, genetic deficiency and targeted inhibition of PAI-1 protect against aging-like pathology and prolong life span. However, the role of PAI-1 in human longevity remains unclear. We hypothesized that a rare loss-of-function mutation in SERPINE1 (c.699_700dupTA), which encodes PAI-1, could play a role in longevity and metabolism in humans. We studied 177 members of the Berne Amish community, which included 43 carriers of the null SERPINE1 mutation. Heterozygosity was associated with significantly longer leukocyte telomere length, lower fasting insulin levels, and lower prevalence of diabetes mellitus. In the extended Amish kindred, carriers of the null SERPINE1 allele had a longer life span. Our study indicates a causal effect of PAI-1 on human longevity, which may be mediated by alterations in metabolism. Our findings demonstrate the utility of studying loss-of-function mutations in populations with geographic and genetic isolation and shed light on a novel therapeutic target for aging.