klotho mutant mice provide a unique model to analyze mechanisms of aging because their phenotypes resemble those of human aging-associated disorders. The klotho gene encodes Klotho, a type I membrane protein that shares sequence similarity with members of the glycosidase family 1. Because Klotho lacks the glutamic acid residues that have been shown to be involved in the catalytic activity of this family of enzymes, the function of this protein was unknown. Here, we have studied the biochemical characteristics of recombinant Klotho. The purified chimeric Klotho-human IgG1 Fc protein (KLFc) was assayed with a series of 4-methylumbelliferyl (4Mu) ␤-glycosides as potential substrates. An enzymatic activity of Klotho was observed only with 4-methylumbelliferyl ␤-D-glucuronide in contrast to bovine liver ␤-glucuronidase, which exhibits a rather wide substrate specificity. Furthermore, the enzymatic activity of KLFc was reduced by the addition of specific inhibitors of ␤-glucuronidase. A number of natural ␤-glucuronides were screened by competitive inhibition for KLFc ␤-glucuronidase. We found that steroid ␤-glucuronides such as ␤-estradiol 3-␤-D-glucuronide, estrone 3-␤-D-glucuronide, and estriol 3␤-D-glucuronide were hydrolyzed by KLFc. The artificial fluorescent substrate and the steroid conjugates share a common phenolic structure. Collectively, these data suggest that Klotho functions as a novel ␤-glucuronidase and that steroid ␤-glucuronides are potential candidates for the natural substrate(s) of Klotho.