An endophytic fungus X-396 was isolated from the leaves of Hypecoum leptocarpum Hook.f.et Thoms. through a tissue separating method.The endophytic fungus X-396 was identified as Diaporthe sp.YG-2015 according to the morphologic observation and ITS sequence analysis.Its fat-soluble components of fermentation were further studied with GC-MS,and antitumor activity was measured by CCK-8 assay.35 kinds of fat-soluble components were detected and identified.The findings showed that the main components of fat-soluble components in fermentation of X-396 were ergosterol (31.83%), β-stitosterol (12.00%) and methyl hexadecanoate (4.22%).Antitumor activity test in vitro showed that the fat-soluble components of fermentation had different degrees of inhibitory effects on human tumor cell lines A549,MCF-7 and HepG2,and HepG2 had the best inhibitory effect with IC₅₀ of 22.65 μg/mL.The results indicate that the fat-soluble components of endophytic fungus X-396 fermentation have the potential to produce antitumor active compounds,and deserve to be deeply investigated.