Increased levels of atmospheric carbon dioxide (CO₂) are likely to affect the trophic relationships that exist between plants, their herbivores, and the herbivores’ natural enemies. We examined interactions across three trophic levels, a transgenic Bacillus thuringiensis Berliner cotton, an aphid herbivore (Aphis gossypii Glover), and a coccinellid predator (Leis axyridis Pallas) as affected by elevated CO₂ concentrations. Experimental CO₂ levels included ambient and 2× and 3× ambient CO₂ levels. Plant height, biomass, leaf area, and carbon:nitrogen (C:N) ratios were significantly higher in plants exposed to elevated CO₂ levels compared with that in plants exposed to ambient CO₂. More dry matter and fat content and less soluble protein were found in A. gossypii in elevated CO₂ treatments. Cotton aphid fecundity significantly increased with increased CO₂ concentrations. The aphid fecundity significantly increased through successive generations reared on plants grown under elevated CO₂, suggesting that the changes in plant quality resulting from elevated CO₂ altered aphid quality as prey for L. axyridis through the food chain. Significantly higher mean relative growth rates were observed in lady beetle larvae under elevated CO₂ treatments. Also, the larval and pupal durations of the lady beetle were significantly shorter and aphid consumption rates increased when fed A. gossypii from elevated CO₂ treatments. Our data showed that increasing CO₂ concentrations could alter the preference of lady beetle to aphid prey and enhance the biological control of aphids by lady beetle. This study provides the first empirical evidence that changes in prey reared on host plants grown at different levels of CO₂ can alter the feeding preference of their natural enemies.