Elevated atmospheric CO₂ concentrations will cause plants to grow faster, lower nitrogen content per unit of plant tissue, and generate higher carbon to nitrogen (C/N) ratios. We hypothesize that production of transgenic proteins will be reduced, thus reducing the efficiency of Bacillus thuringiensis (Bt) transgenes against insect populations. Commercially available transgenic cotton plants expressing the Cry 1Ac gene from Bt were compared with a near isogenic non-Bt cotton line in a split-plot design with two levels of atmospheric CO₂ (ambient, 370 ppm and elevated, 900 ppm) incorporating a 2 × 2 factorial design with two nitrogen (N) fertilization regimes (low, 30 mg N/kg soil/wk and high, 130 mg N/kg soil/wk), and two levels of Bt (presence or absence). Bioassays using Spodoptera exigua (Hübner) and quantitative enzyme-linked immunosorbent assays for toxin content indicated reduced Bt protein production in elevated CO₂. The tendency for test insects to consume more foliage from plants with lower N, caused by the elevated CO₂, did not compensate for the reduction in toxin production. N fertilization regime interacted with CO₂ concentration, showing that plants growing in N limited systems would produce substantially less toxin. The use of transgenic plants is becoming increasingly important and will continue to be so in the next decades. At the same time, atmospheric CO₂ increase will affect the effectiveness of this strategy. These observations have implications not only for agricultural use of transgenic plants, but also for the ecological consequences of transfer of Bt toxins to closely related wild plant genotypes.