This study investigated the antioxidant defense system involved in the tolerance of soybean (Glycine max) to aluminum (Al) stress. Physiological assays showed that the amount of super- oxide free radicals (O2−), hydrogen peroxide (H2O2), and malondialdehyde (MDA) were sig- nificantly lower in an Al-resistant soybean cultivar (cv. PI416937) than in an Al-sensitive soybean cultivar (cv. Huachun18). Comparative analysis of microarray data from both geno- types following Al-stress treatment revealed that the expression of a series of antioxidant enzymes genes was induced in the Al-resistant cultivar. The quantitative real time-PCR (qRT- PCR) assay showed that the transcript levels of genes encoding antioxidant enzymes, including GmCAT1, GmPOD1, GmGST1, GmAPX, GmGSH1, and GmSOD, were higher in the Al-resistant cultivar than in the Al-sensitive cultivar in Al-stress conditions. Furthermore, GmCAT1- overexpressing Arabidopsis plants had improved tolerance to Al-stress and lower O2−, H2O2, and MDA contents than wild-type plants. Therefore, providing evidence that the antioxidant defense system is essential for Al tolerance in soybean.