The expression of double-stranded RNAs (dsRNAs) from the plastid genome has been proven to be an effective method for controlling herbivorous pests by targeting essential insect genes. However, there are limitations to the efficiency of plastid-mediated RNA interference (PM-RNAi) due to the initial damage caused by the insects and their slow response to RNA interference. In this study, we developed transplastomic poplar plants that express dsRNAs targeting the β-Actin (dsACT) and Srp54k (dsSRP54K) genes of Plagiodera versicolora. Feeding experiments showed that transplastomic poplar plants can cause significantly higher mortality in P. versicolora larvae compared with nuclear transgenic or wild-type poplar plants. The efficient killing effect of PM-RNAi on P. versicolora larvae was found to be dependent on the presence of gut bacteria. Importantly, foliar application of a gut bacterial strain, Pseudomonas putida, will induce dysbiosis in the gut bacteria of P. versicolora larvae, leading to a significant acceleration in the speed of killing by PM-RNAi. Overall, our findings suggest that interfering with gut bacteria could be a promising strategy to enhance the effectiveness of PM-RNAi for insect pest control, offering a novel and effective approach for crop protection based on RNAi technology.