Bacillus thuringiensis (Bt) produces vegetative insecticidal proteins (Vip) during its vegetative growth stage. Vip3 proteins have a significant role in insecticidal activity of this bacterium. Each new bacterial isolate may encode Vip with different significance. The Vip protein coding by vip genes of two Bt strains (BnBt and MnD) with high insecticidal activity was characterized in this study. Polymerase chain reaction (PCR)-based screening for Vip genes of these Bt isolates expressed the detected Vip gene and tested the protein for insecticidal activity against the cotton leafworm, Spodoptera littoralis larvae. As a result of the screening tests, Vip3 genes were determined in MnD and BnBt isolates. Vip3 genes of both isolates were expressed and confirmed by 90 kDa proteins. Partially purified and trypsin-activated protein samples of BnBt and MnD isolates were tested against the second instar larvae of S. littoralis. The results showed that the highest insecticidal activity of the Vip3 proteins of BnBt and MnD was 86.66% and 83.33% mortality in 10 days, respectively. The median lethal concentrations (LC50) of BnBt and MnD were determined as 41.860 and 55.154 ng/mu l, respectively. The results suggest that Vip3 protein is effective for preventing resistance in various insect-pest species. The expressed proteins may be utilized as a biopesticide against nocuous insects.