Burrowing nematodes (Radopholus similis) cause severe harm in many agronomic and horticultural crops and are very difficult to manage. Cathepsin S is one of the most important cysteine proteinases and plays key roles in nematodes and many other parasites. To evaluate the effect of in planta RNAi on the control of this nematode, a specific fragment from the protease gene, cathepsin S (Rs-cps), was cloned into the binary vector pFGC5941 in the forward and reverse orientations to construct recombinant plant RNAi vectors. Transgenic Nicotiana benthamiana plants expressing Rs-cps dsRNA were obtained and studied. The transcript abundance of Rs-cps dsRNA appeared to be diverse in the different transgenic lines. Moreover, the bioassay results revealed that Rs-cps transgenic N. benthamiana plants were resistant to R. similis and the transcription level of Rs-cps in R. similis was drastically decreased. In addition, the reproduction and hatching rate of R. similis isolated from the Rs-cps transgenic plants were also significantly reduced. Our results suggest that Rs-cps is essential for the reproduction and pathogenicity of R. similis. This is the first study to employ in planta RNAi approach to target the Rs-cps gene for the control of plant parasitic nematodes.