The 26S proteasome is the major engine of protein degradation in all eukaryotic cells. Adenosine triphosphatase (ATPase) regulatory subunits (Rpts) are constituents of the proteasome that are involved in the unfolding and translocation of substrate proteins into the core particle. In this study, by using the brown planthopper Nilaparvata lugens as a model insect, we report the biological importance of Rpts in female reproduction. We identified six homologous Rpt genes (Rpt1-6) in N. lugens. These genes were detected at high transcript levels in eggs and ovaries of females but at low transcript levels in males. RNA interference-mediated knockdown of N. lugens Rpt genes significantly decreased the proteolytic activity of the proteasome and impeded the transcription of triacylglycerol lipase and vitellogenin genes in the fat bodies and ovaries of adult females and reduced the triglyceride content in the ovaries. The decrease in the proteolytic activity of the proteasome via knockdown of Rpts also downregulated the transcription of the CYP307A2 gene encoding an important rate-limiting enzyme in the 20-hydroxyecdysone biosynthetic pathway in the ovaries, reduced 20E production in adult females and impaired ovarian development and oocyte maturation, leading to the failure of egg production and egg-laying. These novel findings indicate that Rpts are required for the proteolytic activity of the proteasome, which is important for female reproductive success in N. lugens.