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There is an unmet need for treatments for diseases associated with aging. The antiaging, life‐extending, and cognition‐enhancing protein Klotho is neuroprotective due to its anti‐inflammatory, antioxidative, and pro‐myelinating effects. In addition, Klotho is also a tumor suppressor and has beneficial roles in multiple organs. Klotho is downregulated as part of the aging process. Thus, upregulating Klotho in the brain may lead to novel therapeutics to people suffering or at risk for neurodegenerative diseases such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis, and demyelinating diseases such as multiple sclerosis. We attempted to upregulate Klotho for its beneficial effects in the brain and elsewhere. Here, we describe a method to specifically activate Klotho gene expression. To accomplish this task, we designed zinc finger proteins (ZFPs) targeting within −300 bps of the human Klotho promoter. We designed the ZPF constructs either de novo from modular building blocks, or modified sequences from the natural endogenous Egr1 transcription factor backbone structure. Egr1 is known to upregulate Klotho expression. We tested the transcriptional activation effects of these ZFPs in a dual luciferase coincidence reporter system under the control of 4‐kb promoter of human Klotho in stable HEK293 cells and in HK‐2 cells that express Klotho protein endogenously. We found that the best ZFPs are the de novo designed ones targeting −250 bps of Klotho promoter and one of the Egr1‐binding sites. We further enhanced Klotho's activation using p65‐Rta transcriptional activation domains in addition to VP64. These upregulation approaches could be useful for studying Klotho's protective effects and designing Klotho boosting therapeutics for future in vivo experiments. [ABSTRACT FROM AUTHOR]