Background and Purpose: Emerging studies indicated that Klotho is a marker of renal injury and owns a protective effect on different kinds of kidney diseases. However, the role of Klotho in vancomycin (Van)-induced acute kidney injury (AKI) is largely unclear. Hence this study aimed to explore the regulatory mechanism of Klotho in Van-AKI. Experimental Approach: The mRNA expression of Klotho and the JAK2/STAT3/GPx3 axis was assessed by RNA sequence analysis after the Van challenging; the mechanism action of Klotho was examined in vitro by Klotho small interfering RNA and recombinant Klotho. The expression of reactive oxygen species and antioxidant enzymes was detected by flow cytometry and spectrophotometry. Transmission electron microscopy was performed to scan the structural damage of mitochondria. Western blot, qPCR, and immunofluorescence were applied to further explore the function of the JAK2/STAT3/GPx3 axis in Van-AKI. Key Results: RNA sequence analysis indicated that Van challenging reduced the expression of Klotho and GPx3, but increased JAK2/STAT3. In vitro, Klotho siRNA enhanced the production of reactive oxygen species and the cell apoptosis ratio by regulating the JAK2/STAT3/GPx3 axis, while the decrease of GPx3 was prevented by specific inhibitors of JAK2/STAT3. In contrast, recombinant human Klotho showed the opposite function to Klotho siRNA. In vivo, recombinant mouse Klotho improved the anti-oxidative enzyme level and mitochondria damage as well as renal dysfunction and histological damage via upregulating anti-oxidative ability. Conclusion and Implications: To conclude, the study evidenced that recombinant Klotho ameliorated Van-induced AKI via the JAK2/STAT3/GPx3 signaling axis by upregulating anti-oxidative ability.