Brown Planthopper (BPH) causes the most serious damage of the rice crop globally and contributes to the losses of billions of dollars annually. Therefore, RNA interference is an effective gene-silencing tool which makes significant contribution to sustainable agriculture. The present study demonstrated that knockdown of β-1,3-glucanase 5 (Gns5) in a susceptible rice cultivar, TN1 enhanced resistance to BPH. Two RNAi rice genotypes IR463, each using different constitutive promoters IR463 (CaM35S) and IR462 (maize Ubiquitin) were generated, as were their respective empty vector controls. Both RNAi lines significantly (p<0.05) decreased BPH survival and significantly (p<0.05) decreased the rate of development. Survival of insects on the RNAi lines decreased approximately to 50% as compared to their respective transformed empty vector lines and TN1, which were used as controls, with approximately only 30% BPH reaching adulthood on the RNAi lines compared to 75%-80% in the control lines. Microscopic examination showed that callose deposition increased significantly in response to BPH infestation in the sieve plates of the leaf and stem tissues of the IR463 and IR462 lines. In contrast, callose deposition in the control lines was rapidly reduced in response to BPH infestation. This study further revealed that BPH showed a significantly shorter duration of phloem ingestion and higher non-penetration and pathway occurrences in the RNAi lines using EPG studies. In summary, the IR463 and IR462 RNAi lines expressing significantly reduced levels of Gns5 enhanced resistance to BPH, so providing a sound strategy for breeding resistance rice varieties against BPH.