RNA interference (RNAi) is a versatile genetic tool capable of selectively inhibiting the expression of any gene in a targeted organism. Its implementation holds great promise for safeguarding crops against insect pests and diseases. Vacuolar-ATPase represents an ideal target for RNAi-based pest management strategies since it is an enzyme essential for various physiological processes in insects. In this study, double-stranded RNA (dsRNA) was synthesized using an L4440 vector in Escherichia coli HT115 strain to silence the Vacuolar-ATPase proteolipid subunit mRNA in the Colorado potato beetle (Leptinotarsa decemlineata Say). To assess the effectiveness of RNAi, L. decemlineata larvae at different developmental stages were fed potato leaflets treated with dsRNA. The feeding bioassays using dsV-ATPase resulted in significant mortality rates, ranging from 45% to 77% across all of the instar stages of L. decemlineata. Furthermore, ingestion of dsRNAs by third- and fourth-instar larvae exerted significant effects on their body weight and foliage consumption. Notably, feeding the larvae dsV-ATPase led to a significant reduction in V-ATPase gene expression, confirming the efficacy of RNAi-mediated gene silencing in controlling L. decemlineata populations. These findings highlight the potential of RNAi-mediated gene silencing as a valuable strategy for managing L. decemlineata populations by targeting essential genes.