Advances in health care and quality of life in modern societies have led to an increase in the percentage of the population reaching advanced ages. Aging is one of the main risk factors for pathologies including sarcopenia, osteoporosis, and cognitive degeneration. These conditions are accompanied by suffering and elevated economic costs, thus new therapies are needed to achieve healthy aging. The protein Klotho (KL) has been identified as a promising anti-aging molecule due to its beneficial modulation of several molecular pathways altered during aging. Here, we explored the anti-aging potential of the secreted isoform of this protein in the SAMP8 and C57BL mouse models. Systemic and intracerebroventricular delivery of AAV9 efficiently increased concentration of s-KL protein in serum, improving the aging phenotype in different organs analyzed. KL treatment improved fitness in behavioral tests, associated with reduced muscular fibrosis and improved bone microstructural parameters. Cognitive capacities of aged animals were also enhanced, which was accompanied by changes in histological markers like Ki67, DCX, Iba1 and GFAP, supported by transcriptomics analysis. Remarkably, long-term AAV-mediated expression of s-KL lead to a 20% increase in total longevity of C57BL mice. These results show for the first time the pharmacological potential of s-KL expression to reduce simultaneously the impact of age-associated degeneration in multiple organs.