Cleavage and release (shedding) of membrane proteins is a critical regulatory step in many normal and pathological processes. Evidence suggests that the antiaging transmembrane protein Klotho (KL) is shed from the cell surface by proteolytic cleavage. In this study, we attempted to identify the enzymes responsible for the shedding of KL by treating KL-transfected COS-7 cells with a panel of proteinase inhibitors and measuring cleavage products by Western blot. We report that metalloproteinase inhibitors, including EDTA, EGTA, and TAPI-1, inhibit the shedding of KL, whereas insulin increases shedding. The effects of the inhibitors in KL-transfected COS-7 cells were repeated in studies on rat kidney slices ex vivo, which validates the use of COS-7 cells as our model system. Tissue inhibitor of metalloproteinase (Timp)-3 effectively inhibits KL cleavage, whereas Timp-1 and Timp-2 do not, a profile that indicates the involvement of members of the A Desintegrin and Metalloproteinase (ADAM) family. Cotransfection of KL with either ADAM10 or ADAM17 enhances KL cleavage, whereas cotransfection of KL with small interference RNAs specific to ADAM10 and ADAM17 inhibits KL secretion. These results indicate that KL shedding is mediated mainly by ADAM10 and ADAM17 in KL-transfected COS-7 cells. The effect of insulin is abolished when ADAM10 or ADAM17 are silenced. Furthermore, we demonstrate that the effect of insulin on KL shedding is inhibited by wortmannin, showing that insulin acts through a PI3K-dependent pathway. Insulin enhances KL shedding without increasing ADAM10 and ADAM17 mRNA and protein levels, suggesting that it acts by stimulating their proteolytic activities.