The smut fungusUstilago esculentaobligately parasitizesZizania latifoliaand induces smut galls at the stem tips of host plants. Previous research identified a putative secreted protein, Ue943, which is required for the biotrophic phase ofU. esculentabut not for the saprophytic phase. Here, we studied the role of Ue943 during the infection process. Conserved homologs of Ue943 were found in smut fungi. Ue943 can be secreted byU. esculentaand localized to the biotrophic interface between fungi and plants. It is required at the early stage of colonization. TheUe943deletion mutant caused reactive oxygen species (ROS) production and callose deposition in the host plant at 1 and 5 days post inoculation, which led to failed colonization. The virulence deficiency was restored by overexpressing geneUe943orUe943:GFP.Transcriptome analysis further showed a series of changes in plant hormones following ROS production when the host plant was exposed to ΔUe943.We hypothesize that Ue943 might be responsible for ROS suppression or avoidance of recognition by the plant immune system. The mechanism underlying Ue943 requires further study to provide more insights into the virulence of smut fungi.