RNA interference (RNAi) is one of the most promising new approaches for disease therapy. The design of a dumbbell-shaped nanocircular RNA allows it to act as a short interfering RNA (siRNA) precursor. To optimize the design, we studied the relationship between the nanostructure and RNAi activity by synthesizing various RNA dumbbells. An RNA dumbbell with a 23-bp stem and 9-nt loops was the most potent. Sequence analysis by mass spectrometry showed that Dicer could edit RNA dumbbells to siRNA species. The reaction offered the slow release of siRNA species, which conferred prolonged RNAi activity. Introduction of DNA into the loop position significantly stabilized the dumbbell in biological fluid without any loss of RNAi activity. In-depth pharmacological evaluation was performed by introducing dumbbells into HeLa cells that stably express the target luciferase gene. The dumbbells provided a rapid silencing effect and retained this effect for a longer time even at a lower concentration than that at which standard siRNA completely lost RNAi activity. We conclude that an RNA dumbbell with DNA loops is the most promising design for in vivo applications for RNA medicine.