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The antioxidant activity of curcumin has been extensively investigated for years. However, its molecular function mechanism remained unclear. Our preliminary study showed that curcumin downregulated miR-22-3p expression. This study aimed to address whether miR-22-3p mediated the antioxidant activity of curcumin and the possible action mechanism. The results showed that miR-22-3p repression increased the total cellular antioxidant capacity and enhanced the endogenous antioxidant enzyme system of LO2 cells. In addition, miR-22-3p-deteriorated cellular antioxidant capacity was prevented by curcumin pretreatment. Moreover, miR-22-3p inhibition significantly improved mitochondrial function and biogenesis. Further investigation demonstrated that a mitochondrial key regulator, the cardiolipin synthase gene (CRLS1), was targeted and downregulated by miR-22-3p. CRLS1 overexpression also significantly improved cellular antioxidant capacity and enhanced mitochondrial function and biogenesis in LO2 cells. Taken together, all these data suggested that miR-22-3p modulated the antioxidant effect of curcumin via targeting CRLS1, which may provide novel insights into miRNA-mediated antioxidant mechanism of curcumin.