Malate transporters play a critical role in aluminum (Al) tolerance responses for some plant species, such as Arabidopsis (Arabidopsis thaliana). Here, we further characterize AtALMT1, an Arabidopsis aluminum-activated malate transporter, to clarify its specific role in malate release and Al stress responses. Malate excretion from the roots of accession Columbia was sharply induced by Al, which is concomitant with the induction of AtALMT1 gene expression. The malate release was specific for Al among rhizotoxic stressors, namely cadmium, copper, erbium, lanthanum, sodium, and low pH, which accounts for the specific sensitivity of a null mutant to Al stress. Al-specific malate excretion can be explained by a combined regulation of AtALMT1 expression and activation of AtALMT1 protein, which is specific for Al. Although low pH treatment slightly induced gene expression, other treatments did not. In addition, malate excretion in Al-activated seedlings was rapidly stopped by removing Al from the solution. Other rhizotoxic stressors were not effective in maintaining malate release. Protein kinase and phosphatase inhibitor studies indicated that reversible phosphorylation was important for the transcriptional and posttranslational regulation of AtALMT1. AtALMT1 promoter-beta-glucuronidase fusion lines revealed that AtALMT1 has restricted expression within the root, such that unnecessary carbon loss is likely minimized. Lastly, a natural nonsense mutation allele of AtALMT1 was identified from the Al-hypersensitive natural accession Warschau-1.