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The aim of this study was to investigate the anti-ageing effect of Yisui moxibustion on regulating the klotho protein and insulin (INS)/IGF-1 signalling cascade in d-galactose mice. Sixty male Kunming mice were randomly divided into five groups after the Morries test: the normal sodium, model, moxa cone moxibustion 1 and 2, and sham groups. The treatment and model were given d-galactose injection for 70 days, while mice in the normal sodium group were injected the same dosage of normal sodium. From day 13 after the first injection, the three treatment groups were treated every day for 58 days. The INS and klotho in the cerebral tissues were measured. p-Akt, p-FoxO3a, Klotho, SOD2 expression were observed immunohistochemically. The escape latency of mice in the model and sham were significantly prolonged, and the spanning original platform times were significantly decreased compared with the normal sodium and moxa cone moxibustion 1 and 2 (p < 0.01). The content of INS and klotho in the model was significantly lower than the normal sodium, moxa cone moxibustion 1 and 2 (p < 0.01). Compared with the model and sham, p-Akt and p-FoxO3a expression in moxa cone moxibustion 1 and 2 were markedly decreased (p < 0.05), and the expressions of klotho and SOD2 were significantly increased (p < 0.05) in hippocampal CA1. Yisui moxibustion could exert the antioxidative effect on klotho protein, which negatively regulated the insulin signalling pathway and its downstream elements in cerebral tissue and hippocampal CA1, so as to enhance learning and memory capacities, delay brain ageing. [ABSTRACT FROM AUTHOR]