The problem of heavy metal contamination in soil is becoming more and more serious due to the booming industrial development and the increasing pollution from domestic waste, and the excessive concentration of heavy metals in crops will eventually seriously threaten human health. Aluminum-activated malate transporters (ALMT) encode a class of anion channel proteins that play important roles in the transmembrane transport of plant organic acids. In order to investigate the function of GmALMT33 gene in response to Cd stress in soybean, the GmALMT33 gene was cloned from soybean using RT-PCR in this study. Bioinformatics analysis showed that the CDS region of the gene is 1622 bp in length, encodes 553 amino acids, contains the ALMT structural domain and five transmembrane structural domains, and the GmALMT33 protein is evolutionarily closest to the homologous proteins in kidney bean. qRT-PCR was performed to investigate the relative expression levels of the GmALMT33 gene in different tissues of soybean and after cadmium stress. The expression level of GmALMT33 was highest in soybean roots; after cadmium stress, the expression of this gene showed a trend of first increasing and then decreasing. We constructed the plant expression vector pCPB-GmALMT33 and genetically transformed tobacco and soybean haiey roots. Phenotypic analysis of the transgenic plants showed that under cadmium stress (66 mmol/L CdCl2), the leaf blades of the transgenic tobacco were yellowed and greenish, and the degree of browning at the edges was significantly lower than that of wild-type tobacco. The transgenic soybean hairy root complex plants showed significantly weaker degree of reddish-brown toxicity symptoms in stalks and leaf veins than the trans-space vector plants. After 0 d, 1 d and 3 d of cadmium stress treatment, the SOD, APX activity and soluble sugar content of roots and leaves of transgenic soybean hairy root complex were higher than those of the trans-space vector control, and the MDA content was lower than that of the control; after 7 d of cadmium stress treatment, the SOD, APX activity and soluble sugar content of transgenic tobacco leaves were higher than those of the wild-type control, and the MDA content was lower than that of the control, which indicated that the GmALMT33 gene improved the toxicity symptoms of transgenic soybean hairy root complex. GmALMT33 gene improved the cadmium tolerance ability of plants. This study provides a basis for further exploring the mechanism of action of GmALMT33 gene and provides a new gene for soybean stress tolerance breeding.