Agricultural pest infestation is as old as domestication of food crops and contributes a major share to the cost of crop production. In a transgenic pest control approach, plant production of Vip3A, an insecticidal protein from Bacillus thuringiensis, is effective against lepidopteran pests. A synthetic Vip3A gene was evaluated for efficacy against Spodoptera litura Fabricius (Lepidoptera: Noctuidae; cotton leafworm), Spodoptera exigua Hübner (Lepidoptera: Noctuidae; beet armyworm), Spodoptera frugiperda Smith (Lepidoptera: Noctuidae; fall armyworm), Helicoverpa armigera Hübner (Lepidoptera: Noctuidae; cotton bollworm), Helicoverpa zea Boddie (Lepidoptera: Noctuidae; corn earworm), Heliothis virescens Fabricius (Lepidoptera: Noctuidae; tobacco budworm), and Manduca sexta L. (Lepidoptera: Sphingidae; tobacco hornworm) in tobacco. In artificial diet assays, the concentration required to achieve 50% mortality was highest for H. zea followed by H. virescens > S. exigua > H. armigera > M. sexta > S. frugiperda > S. litura. By contrast, in bioassays with detached leaves from Vip3A transgenic tobacco, the time until 50% lethality was M. sexta > H. virescens > S. litura > H. zea > H. armigera > S. exigua. There was no significant correlation between the artificial diet and transgenic plant bioassay results. Notably, the two insect species that are best-adapted for growth on tobacco, M. sexta and H. virescens, showed the greatest time to 50% mortality on Vip3A-transgenic tobacco. Together, our results suggest that artificial diet assays may be a poor predictor of Vip3A efficacy in transgenic plants, lepidopteran species vary in their sensitivity to Vip3A in diet-dependent manner, and host plant adaptation of the targeted herbivores should be considered when designing transgenic plants for pest control.