Objective To study the antitumor natural products from Berlandiera lyatra. Methods Compounds were isolated by liquid/liquid partition and chromatography on Sephadex LH20 and silica gel columns. All process of the fractionation were run with the guidance of cytotoxicity bioassay by MTT method against three human cancer cell lines. The chemical structures of bio-active compounds were identified on the basis of spectroscopic data. Results From the CHCl3-soluable fraction of the title plant, two cytotoxic compounds were isolated. They were determined as 3α-epoxypumilin and pumilin. Conclusion 3α-epoxypumilin and pumilin were responsible major compounds for the cytotoxicity in the methanolic extract of the plant. Both compounds were isolated from the B. lyatra and were reported to be of antitumor activity for the first time.