Toxic aluminum enters the root cells rapidly, therefore internal detoxification is required. However, the molecular mechanisms underlying this process are poorly understood. Here we functionally characterized a rice gene, Os03g0755100 (OsALS1), that is regulated by ART1, a C2H2-type zinc finger transcription factor.OsALS1encodes a half-size ABC transporter that is a member of the TAP (transporter associated with antigen processing) sub-group. Expression ofOsALS1was rapidly and specifically induced by Al in the roots, but not by other metals or low pH. OsALS1 was localized at all cells of the roots. Furthermore, OsALS1 is localized to the tonoplast. These expression patterns and cell specificity of localization are different from those of the homologous geneAtALS1in Arabidopsis. Knockout ofOsALS1in three independent lines resulted in significant increased sensitivity to Al, but did not affect the sensitivity to other metals and low pH. Comparison of Al accumulation patterns between wild-type andosals1mutants showed that there was no difference in Al levels in the cell sap of root tips between wild-type and the mutants, but the mutants accumulated more Al in the cytosol and nucleus than the wild-type. Expression ofOsALS1in yeast resulted in increased Al sensitivity due to mis-localization. These results indicate that OsALS1 localized at the tonoplast is responsible for sequestration of Al into the vacuoles, which is required for internal detoxification of Al in rice.