We report here the identification of mouse βklotho (βkl), which encodes a type I membrane protein with high resemblance to Klotho (KL). Both βKL and KL consist of two internal repeats with homology to family 1 glycosidases, while these essential glutamates for the enzymatic activities were not conserved. The identical pattern of substitution and variation in the substituted amino acids between these two proteins indicate that they likely to form a unique family within the glycosidase family 1 superfamily. During mouse embryonic development, strong βkl expression was detected in the yolk sac, gut, brown and white adipose tissues, liver and pancreas, and in the adult, predominantly in the liver and pancreas. Despite the high structural similarity between βKL and KL, their expression profiles were considerably different and βkl expression was not induced in kl-deficient mouse mutants.