The gene Klotho encodes a type-I-membrane protein expressed in two forms, membrane and secreted. Inhibition of klotho induces an accelerated age phenotype whereas its over-expression extends lifespan and thus has been proposed as a promising candidate for treating age-related disorders. In the last years we have been focused on gene therapy strategies with klotho as a therapy for cognitive deficits associated with aging and neurodegeneration. Our studies demonstrated that just a single in vivo administration of AAVs expressing soluble klotho (sKL) resulted in long-lasting enhancement memory in non-pathological aged and accelerated-senescence (SAMP8) mice. Also, sKL increases bone density, reduces bone fragility and osteopenia, as well as increases muscle strength and reduces sarcopenia. Moreover, long-term expression of sKL increases longevity and rejuvenate the neuronal epigenetic profile. Considering all these data, in the present study we investigate the effects of gene-therapy with sKL in Alzheimer’s disease (AD). Principal results showed that sKL overexpression tended to improve spatial learning and reference memory in the MWM in males as well as the recognition and spatial memory in the NOR and T-maze tests in females of the APP/Tau model. In this context, and with the objective to get closer to humans, we determined that sKL is the predominant klotho transcript expressed in human brains, both in healthy-aged and AD’s patients. Furthermore, sKL therapy is a safety strategy and improves cognitive performance in aged non-human primates. In conclusion, we demonstrate that sKL is an attractive therapy with high potentiality to treat human brain pathologies like AD.