AbstractRNA interference (RNAi) is a technique used in many insects to study gene function. However, prior research suggests possible off-target effects when usingGreen Fluorescent Protein(GFP) sequence as a non-target control. We used a transcriptomic approach to study the effect ofGFPRNAi (GFP-i) inNasonia vitripennis, a widely used parasitoid wasp model system. Our study identified 3.4% of total genes being differentially expressed in response toGFP-i. A subset of these genes appears involved in microtubule and sperm functions.In silicoanalysis identified 17 potential off-targets, of which only one was differentially expressed afterGFP-i. We suggest the primary cause for differential expression afterGFP-i is the non-specific activation of the RNAi machinery at the injection site, and a potentially disturbed spermatogenesis. Still, we advise that any RNAi study involving the genes deregulated in this study, exercises caution in drawing conclusions and uses a different non-target control.