Effectors are crucial for the interaction between endophytes and their host plants. However, limited attention has been paid to endophyte effectors, with only a few reports published. This work focuses on an effector ofFusarium lateritium, namelyFlSp1(Fusarium-lateritium-Secreted-Protein), a typical unknown secreted protein. The transcription ofFlSp1was up-regulated after 48 h following fungal inoculation in the host plant, i.e., tobacco. The inactivation ofFlSp1with the inhibition rate decreasing by 18% (p< 0.01) resulted in a remarkable increase in the tolerance ofF. lateritiumto oxidative stress. The transient expression ofFlSp1stimulated the accumulation of reactive oxygen species (ROS) without causing plant necrosis. In comparison with the wild type ofF.lateritium (WT), theFlSp1mutant of theF. lateritiumplant (ΔFlSp1) reduced the ROS accumulation and weakened the plant immune response, which resulted in significantly higher colonization in the host plants. Meanwhile, the resistance of the ΔFlSp1plant to the pathogenicRalstonia solanacearum, which causes bacterial wilt, was increased. These results suggest that the novel secreted proteinFlSp1might act as an immune-triggering effector to limit fungal proliferation by stimulating the plant immune system through ROS accumulation and thus balance the interaction between the endophytic fungi and their host plants.