Background: UV-related skin disease such as actinic keratosis is a major concern in public health. In view of the cell injury induced by UVB, Klotho protein it is an ideal therapy to eliminate UVB-induced cell damages and the associated signaling pathways. Material/Methods: To gain insights into the potential role of Klotho and the underlying molecular mechanism, we constructed a Klotho-overexpress HaCaT cell line and assessed the protection against UVB insults. The effects of exposure to UVB radiation on the human keratinocyte HaCaT cells, including cell growth, apoptosis, and changes of selected biomarkers, were measured by CCK-8, flow cytometry, Quantitative real-time PCR, and Western blot analysis. Results: We found that enhanced NE-kappa B activity was accompanied by decreased expression of the anti-aging protein Klotho upon UVB stimulation, which was further confirmed with in vivo experiments. Overexpression of Klotho was able to considerably alleviate the UVB-induced damages to cells and reversed the UVB-caused biomarker changes to a great extent, which was comparable to the effects of administration of NE-kappa B inhibitor PDTC, suggesting the inhibition of nuclear translocation and DNA-binding activity of NF-kappa B. Furthermore, Klotho overexpression was proved to decrease the nuclear expression of NE-kappa B as much as the treatment with PDTC, which provides support for the direct regulation of NE-kappa B by Klotho. Conclusions: Collectively, our work provides new insight into the potential role of Klotho in the context of UVB-induced inju- ries in human keratinocytes, as well as providing the basis for future study of new therapies against UV-related skin disease.