The secondary metabolites of Fusarium sporotrichioides , an endophytic fungus with anti-tumor activity isolated from Rauvolfia yunnanensis Tsiang, were investigated. Five trichothecenes, including one previously undescribed metabolite, were isolated and identified. Their structures were elucidated by means of extensive spectroscopic methods; the absolute configuration of compound 1 was determined by the ECD method. Surprisingly, 8-n-butyrylneosolaniol ( 3 ) exhibited stronger anti-tumor activity than T-2 toxin against Huh-7 cell line, with an IC 50 value of 265.9 nM. 8-n-butyrylneosolaniol ( 3 ) promoted apoptosis induction in Huh-7 cells. Moreover, cell cycle analysis showed that cell cycle arrest caused by 8-n-butyrylneosolaniol ( 3 ) at the G2/M phase resulted in cell proliferation inhibition and pro-apoptotic activity. Further studies showed a significant decrease in mitochondrial membrane permeabilization and a significant increase in ROS generation, which led to the activation of caspase cascades and subsequent cleavage of PARP fragments. In conclusion, 8-n-butyrylneosolaniol ( 3 ) induced cell apoptosis in Huh-7 cells via the mitochondria-mediated apoptotic signaling pathway, which could be a leading compound for anti-tumor agents.