Betaine (BET) is a native compound widely studied as an antioxidant in agriculture and human health. However, the antioxidant mechanism of BET remains unclear. In this research, radical scavenging assays showed that BET had little free radical scavenging activity. However, the antioxidant activity of BET was confirmed by cellular antioxidant activity (CAA) and erythrocyte hemolysis assays. The results of quantitative PCR (qPCR) and enzyme activity determination kits showed that the antioxidant activity of BET was not due to the gene expression and activity of antioxidases. High-pressure liquid chromatography (HPLC) assessment of the effect of BET on sulfur-containing amino acid metabolism showed that BET increased the levels of nonenzymatic antioxidants,S-adenosylmethionine (SAM) and methionine (p < 0.05), via the regulation of the methionine–omocysteine cycle. Additionally, the three methyl groups of BET were found to play a key role in its antioxidant activity. The possible reason was that because of the hydrophobicity of the three methyl groups and hydrophilicity of the carboxyl of BET, a tight protective membrane was formed around cells to prevent oxidative stress inducer from inducing ROS generation and cell damage. In conclusion, the antioxidant mechanism of BET was found to enhance nonenzymatic antioxidant defenses via the methionine–homocysteine cycle and form a protective membrane around cells.