Aluminum (Al) stress represses mitochondrial respiration and produces reactive oxygen species (ROS) in plants. Mitochondrial alternative oxidase (AOX) uncouples respiration from mitochondrial ATP production and may improve plant performance under Al stress by preventing excess accumulation of ROS. We tested respiratory changes and ROS production in isolated mitochondria and whole cell of tobacco (SL, ALT 301) under Al stress. Higher capacities of AOX pathways relative to cytochrome pathways were observed in both isolated mitochondria and whole cells of ALT301 under Al stress. AOX1 when studied showed higher AOX1 expression in ALT 301 than SL cells under stress. In order to study the function of tobacco AOX gene under Al stress, we produced transformed tobacco cell lines by introducing NtAOX1 expressed under the control of the cauliflower mosaic virus (CaMV) 35 S promoter in sensitive (SL) Nicotiana tabacum L. cell lines. The enhancement of endogenous AOX1 expression and AOX protein with or without Al stress was in the order of transformed tobacco cell lines > ALT301 > wild type (SL). A decreased respiratory inhibition and reduced ROS production with a better growth capability were the significant features that characterized AOX1 transformed cell lines under Al stress. These results demonstrated that AOX plays a critical role in Al stress tolerance with an enhanced respiratory capacity, reducing mitochondrial oxidative stress burden and improving the growth capability in tobacco cells.