Background: Most recently, major federal regulatory agencies deregulated an in planta RNA interference (RNAi) trait against a devastating corn pest, the western corn rootworm Diabrotica virgifera virgifera, in the United States and Canada. The impact of double-stranded RNA (dsRNA) plant-incorporated protectants (PIPs) and dietary RNAi to non-target organisms, however, still needs further investigation. In this study, we assessed the potential risks of a Diabrotica virgifera virgifera active dsRNA to a group of predatory biological control agents, including Hippodamia convergens, Harmonia axyridis, Coleomegilla maculata, and Coccinella septempunctata. The overarching hypothesis is that the insecticidal dsRNA targeting Diabrotica virgifera virgifera has no or negligible adverse effect on lady beetles. Results: A 400-bp fragment with the highest sequence similarity between target and tested species was selected as the template for dsRNA synthesis. For the dietary RNAi toxicity assay, newly hatched first instar larvae were administered with v-ATPase A dsRNAs designed from Diabrotica virgifera virgifera and the four lady beetles, respectively. A dsRNA from ⊎-glucuronidase (GUS), a plant gene, and H2O were served as the negative controls. The endpoint included both sub-organismal (gene expression), and organismal (survival rate, development time, pupa and adult weight) measurements. The results from dietary RNAi toxicity assay demonstrate significantly impacts of Diabrotica virgifera virgifera-active dsRNAs on lady beetles under the worstcase scenario at both transcriptional and phenotypic level. Interestingly, substantial differences among the four lady beetle species were observed toward the ingested exogenous dsRNAs. Conclusion: Such differential response to dietary RNAi may shed light on the mechanisms underlying the mode-of-action of RNAi-based biopesticides