Abstract: Sugarcane is the most important sugar crop in the world. Sugarcane smut caused by Sporisorium scitamineum is a worldwide disease seriously affecting the sugar yield. RNA interference（RNAi） technology is a rapid and effective tool for studying gene function in plant. However, there has been no report on RNAi in S. scitamineum. In this study, the enhanced green fluorescent protein（eGFP） was used as a test target gene. We constructed JG35-eGFP strain expressing eGFP and JG36-eGFP-RNAi strain containing palindrome sequences of eGFP gene, respectively. After fusion of the hypae of the two transformants by mating, it was observed that the eGFP gene expression and the fluorescence intensity were significantly reduced, demonstrating the feasibility of RNA interference in S. scitamineum. We then used this interference system to study the function of the effector gene g2476. It was found that the gene g2476 was silenced in both haploid sporidium and dikaryotic hyphae but the silencing strains were indistinguishable to the wild-type strain in mating/filamentation, suggesting that this effector might not involved in mating/filamentation. Thus, this RNAi method provides another avenue for the study of gene function for S. scitamineum, and for breeding HIGS（Host induced gene silencing） disease-resistant plants.