Klotho protein is well-known as an anti-aging agent, however, several studies have suggested that Klotho protein also increases antioxidant activity and the reproductive system, as Klotho protein is closely associated with Wnt signaling. The objective of our study was to investigate the enhancement of porcine oocyte in vitro maturation via the Klotho protein-Wnt signaling pathway. Following immunohistochemistry and ELISA, we treated cells with Klotho protein during in vitro maturation. Lithium Chloride, a specific activator of Wnt signaling, was subsequently co-administered with Klotho protein. Mature oocytes subjected to treatments were used for the analysis of embryonic development, qRT-PCR, and immunocytochemistry. Treatment with 5pg/ml Klotho protein significantly increased cumulus cell expansion, blastocyst formation rates, and the total cell number of blastocysts. During cotreatment with 5mM Lithium Chloride and 5pg/ml Klotho protein, blastocyst formation rates were the highest in Klotho protein-treated oocytes and the lowest in Lithium Chloride-treated oocytes. Expression levels of Wnt signaling-related transcripts and proteins were significantly impacted by Klotho protein and Lithium Chloride. Moreover, cellular ATP levels and antioxidant activities were enhanced by Klotho protein treatment. These findings suggest a significant involvement of the Klotho protein-Wnt signaling mechanism in porcine oocyte maturation.