Aging is a major risk factor for many neurodegenerative diseases. Klotho (KL) is a glycosylated transmembrane protein that is expressed in the choroid plexus and neurons of the brain. KL exerts potent anti-aging effects on multiple cell types in the body but its role in human brain cells remains largely unclear. Here we show that human cortical neurons, derived from human pluripotent stem cells in 2D cultures or in cortical organoids, develop the typical hallmarks of senescent cells when maintained in vitro for prolonged periods of time, and that moderate upregulation or repression of endogenous KL expression in cortical organoids inhibits and accelerates senescence, respectively. We further demonstrate that KL expression alters the expression of senescence-associated genes including, extracellular matrix genes, and proteoglycans, and can act in a paracrine fashion to inhibit neuronal senescence. In summary, our results establish an important role for KL in the regulation of human neuronal senescence and offer new mechanistic insight into its role in human brain aging.