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Secretion and efflux of oxalic acid from roots is an important aluminum detoxification mechanism for various plants; however, how this process is completed remains unclear. In this study, the candidate oxalate transporter gene <i>AtOT</i>, encoding 287 amino acids, was cloned and identified from <i>Arabidopsis thaliana</i>. <i>AtOT</i> was upregulated in response to aluminum stress at the transcriptional level, which was closely related to aluminum treatment concentration and time. The root growth of <i>Arabidopsis</i> was inhibited after knocking out <i>AtOT</i>, and this effect was amplified by aluminum stress. Yeast cells expressing <i>AtOT</i> enhanced oxalic acid resistance and aluminum tolerance, which was closely correlated with the secretion of oxalic acid by membrane vesicle transport. Collectively, these results underline an external exclusion mechanism of oxalate involving <i>AtOT</i> to enhance oxalic acid resistance and aluminum tolerance.