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RNA interference (RNAi) technology uses dsRNAs to silence specific targeted genes by downregulating their expression. It has become a potent tool for functional and regulatory studies of insect genes and has potential to be applied for insect control. Though it has been challenging to generate effective RNAi in lepidopteran insects, in the current study this technology was applied to develop specific RNAi-based molecular tools that could be used to negatively impact the invasive lepidopteran forest pest, gypsy moth (GM). GM midgut-specific genes were selected for dsRNA design from larval transcriptome profiles. Two methods were used to produce specific dsRNAs, bacterial expression and in vitro synthesis, which were then fed per os to GM larvae. Depletion of uncharacterized gene targets known as locus 365 and locus 28365, or their stacked combination, depleted target transcripts in a sequence specific manner and resulted in 60% reduction in body mass. Treated GM females that were able to moult to the adult stage displayed an approximately two-fold reduction in egg masses. These have potential to be developed as molecular biopesticides for GM.