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Aluminum (Al) and cadmium (Cd) often coexist in the acidic soils of southern China, but their interactive effects remain unclear. In this study, this was examined in Al-resistant (Atlas 66) and Al-sensitive (Scout 66) wheat cultivars. The results showed that Al significantly alleviated the root growth of Al-tolerant Atlas 66 under Cd stress but had no effect on the root growth of Al-sensitive Scout 66 or the shoot growth of both cultivars. However, Al substantially decreased Cd accumulation in the roots and shoots of Atlas 66 and Scout 66. This could be attributed to the decreased uptake of Cd by the roots rather than the changes in Cd distribution in various organs or the Cd binding and adsorption of the cell wall. Using non-invasive micro-test technology, we further confirmed that Al and Cd co-exposure significantly inhibited the net Cd2+ influx into the roots of Atlas 66 and Scout 66 compared to Cd-only stress. Furthermore, the higher Cd2+ influx into roots should be responsible for the high Cd accumulation in the roots and shoots of Atlas66 than in those of Scout66 under Al and Cd co-exposure, which may be due to the higher Al accumulation in the roots of Al-sensitive Scout 66 that resulted in the larger decrease of negative charges on root surfaces. Finally, the effects of Al on the expression of Cd-related genes responsible for Cd uptake and translocation in wheat roots were investigated. The results have suggested that Al significantly downregulated the expression of TaNramp5 in Atlas 66 and the expression of TaNramp5 , TaIRT1 and TaHMA2 in Scout66. • Al led to a significant decrease of Cd accumulation in wheat plants. • Al decreased the Cd uptake by wheat roots. • A lower Cd2+ influx into roots was detected in Al-sensitive Scout 66. • Al significantly down-regulated the expressions of Cd-related genes in wheat roots. [ABSTRACT FROM AUTHOR]