Trichoderpyrone (1), a unique polyketide with a cyclopentenone-pyrone hybrid skeleton, was isolated from the plant endophytic fungus Trichoderma gamsii. The structure of 1 was determined by detailed analysis of NMR data together with comparison of chemical shift values of similar fragments. The relative and absolute configurations were established by NOESY correlations and CD experiment. Trichoderpyrone (1) displayed weak cytotoxic activities against A549, HepG2, and HeLa cancer cell lines. 1 might originate from a hybrid biosynthetic pathway through two nonreduced (NR) polyketide megasynthetases.