In order to explore the chemical constituents and its cytotoxic activity of stems and leaves of Nicotiana rustica L. from Yunnan,12 compounds were isolated from the 95% ethanol extracts of the sample by using macroporous resin column chromatography,silica gel column chromatography,MCI column chromatography and Sephadex LH-20 gel column chromatography.Through the analysis of spectral data and references,their structures were identified as anatabin (1),trans-N-coumaroyltyramine (2),N-trans-feruloyltyramine (3),N-trans-caffeoyltyramine (4),9-epi-blumenol C (5),(2R,3S)-［2,3,4,4-²H₄］-Hexadecanoic acid (6),trans-phytyl palmitate (7),oleanolic acid (8),cis-abienol (9),ursolic acid (10),dihydroactinidiolide (11),(1S,2E,4R,6E,8R,11S)-8,11-epoxy-2,6,12(20)-cembratrien-4-ol (12).Compounds 5-7 were isolated from the Nicotiana genus for the first time.The effects of compounds 1-12 on human hepatoma cells SMMC-7721,Hep3β and human colon cancer LOVO cells were determined by CCK-8 method.Compounds 1-4,8-10 and 12 showed definite antitumor activity.Among them,compound 10 has a significant effect on human liver cancer Hep3β and human colon cancer LOVO cells.The inhibition rate of cells was significantly higher than that of positive control drugs,the IC₅₀ values were 48.21 μmol/L and 87.34 μmol/L,respectively.