[Objectives]  Destruxins are insecticidal secondary compounds isolated from Metarhizium anisopliae. In this study, the toxicity of different destruxins was tested on four insect cell lines, i.e., Bombyx mori Bm12, Ostrinia furnacalis hemocytes Ofh, Drosophila melanogaster S2 and Spodoptera frugiperda Sf9. [Methods]  MTT and microscopy analysis were employed to evaluated the effects of destruxins A and B (DA and DB), and a 1/1 (m/m) mixture of these compounds (DABM), on the four insect cell lines. Relative toxicity was assessed by comparing IC₅₀s values and morphological changes in the cell lines. [Results]  The results indicate that Ofh cells were more susceptible to lower dosages (< 25 μg/mL) of DA and DB, with IC₅₀ values of 219.19, 112.29 and 34.86 μg/mL for DA, DB and DABM, respectively, compared to IC₅₀ values > 250 μg/mL in the other cell types. Furthermore, DA and DB had a synergistic effect on Ofh cells, but an antagonistic effect on Sf9 cells, and an additive effect on Bm12 and S2 cells. Microscopy revealed morphological changes in the different cell lines, even at dosages as low as 6.25 μg/ mL. Moreover, these morphological changes became more apparent at higher dosages. Ruptured cells, strumae, aggregation, expanded cells, vacuoles and reduced cell numbers were found in Bm12 cells. In Ofh cells, cells became expanded but appeared to return to normal with aggregation. Vacuoles and a few expanded, ruptured and aggregated cells were found in the S2 cell line, and membrane shrinkage, broken cells and a decrease in cell numbers were recorded in the Sf9 cell line. [Conclusion]  The Ofh cell line was the most sensitive to destruxins, and the non-host insect Drosophila melanogaster S2 cell line the least sensitive. The results suggest that although relatively large doses of destruxins are required to kill insect cells, smaller dosages are sufficient to cause changes in cell morphology.