Aim To investigate the effect and mechanism of Klotho on lipopolysaccharide(LPS) induced cardiomyocyte damage. Methods H9c2 cells were pretreated with different concentrations of Klotho protein, then treated with 1 g/L LPS for 6 hours, and cardiomyocyte damage was estimated by detecting the content of lactate dehydrogenase(LDH) and cell viability. Inflammation was estimated by detecting the content of tumor necrosis factor-α (TNF-α), interleukin-β(IL-β), IL-6. The status of oxidative stress was measured by malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px). Cell apoptosis was measured with flow cytometry. The expression of nuclear factor-κB (NF-κB)p65 and p-NF-κB p65 at protein levels were measured with Western blot assay. Results Klotho can alleviate LPS-induced decrease of cell viability, inhibit the release of LDH, TNF-α, IL-β, IL-6, down-regulate the content of MDA, enhance the activity of SOD, GSH-Px, suppress cell apoptosis and p-NF-κB p65 protein expression (P<0.05). Conclusion Klotho can alleviate LPS-induced cardiomyocyte damage. And its mechanism may be related to the downregulation of NF-κB activation, and protect H9c2 cells from LPS-induced inflammation, oxidative stress and cell apoptosis.