The Klotho protein is a β-glucuronidase, and its overexpression is associated with life extension. Its mechanism of action is not fully understood, although it has been recently reported that αKlotho improves synaptic and cognitive functions, and it may also influence a variety of structures and functions during CNS maturation and aging. The αKlotho gene has two transcripts, one encoding a transmembrane isoform (m-KL), and the other a putative secreted isoform (s-KL). Unfortunately, little is known about the secreted αKlotho isoform, since available antibodies cannot discriminate s-KL from the KL1 domain cleaved from the transmembrane isoform. This study shows, for the first time, that the klotho transcript produced by alternative splicing generates a stable protein (70 kDa), and that in contrast to the transmembrane Klotho isoform, it is ten times more abundant in the brain than in the kidney suggesting that the two isoforms may have different functions. We also studied whether klotho expression in the CNS was influenced by aging, Alzheimer's disease (AD), or a healthy lifestyle, such as voluntary moderate continuous exercise. We observed a strong correlation between high expression levels of the two klotho transcripts and the healthy status of the animals. Expression of Klotho in brain areas decayed more rapidly in the 3xTg-AD model of AD than in healthy animals, whilst moderate continuous exercise in adulthood prevents the decline in expression of both klotho transcripts.