Aluminum (Al) solubilizes into trivalent ions (Al(3+)) on acidic soils, inhibiting root growth. Since about 13% of global rice cultivation is grown on acidic soils, improving Al tolerance in rice may significantly increase yields. In the present study, metabolome analysis under Al toxicity between the Al-tolerant variety Nipponbare and the Al-sensitive variety H570 were performed. There were 45 and 83 differential metabolites which were specifically detected in Nipponbare and H570 under Al toxicity, respectively. Furthermore, the results showed that 16 lipids out of 45 total metabolites were down-regulated, and 7 phenolic acids as well as 4 alkaloids of 45 metabolites were up-regulated in Nipponbare, while 12 amino acids and their derivatives were specifically detected in H570, of which 11 amino acids increased, including L-homoserine and L-methionine, which are involved in cysteine synthesis, L-ornithine and L-proline, which are associated with putrescine synthesis, and 1-aminocyclopropane-1-carboxylate, which is associated with ethylene synthesis. The contents of cysteine and s-(methyl) glutathione, which were reported to be related to Al detoxification in rice, decreased significantly. Meanwhile, putrescine was accumulated in H570, while there was no significant change in Nipponbare, so we speculated that it might be an intermediate product of Al detoxification in rice. The differential metabolites detected between Al-tolerant and -sensitive rice variants in the present study might play important roles in Al tolerance. These results provide new insights in the mechanisms of Al tolerance in rice.