Summary Virus‐derived small interfering RNAs (vsiRNAs) play an important role in viral infection by regulating the expression of host genes. At present, research on the regulation of plant primary metabolic pathways by vsiRNAs is very limited. TvsiRNA24 derived from tobacco curly shoot virus (TbCSV) was amplified by reverse transcription polymerase chain reaction, and its target gene NbTPI (triosephosphate isomerase) was verified using reverse transcription quantitative polymerase chain reaction and GFP fluorescence observation. The effect of the interaction between TvsiRNA24 and NbTPI on TbCSV infection was analyzed by virus mediated, genetic transformation, western blotting, and quantitative detection. The expression of TvsiRNA24 retards the growth of Nicotiana benthamiana and enhances TbCSV accumulation within N. benthamiana. The overexpression of NbTPI attenuates the accumulation of TbCSV, and the silencing of NbTPI leads to the growth retardation of N. benthamiana and intensifies symptoms post‐TbCSV infection. Moreover, the expression of some genes related to photosynthesis, primary metabolism and immune response is regulated by NbTPI. Our results unveil the specific role of TvsiRNA24‐NbTPI in the pathogenicity of TbCSV, resulting in hindrance to plant growth and facilitation of viral infection. The identification of this regulatory pathway provides valuable insights that can be utilized to devise novel antiviral approaches targeting the reduction of viral pathogenicity.