In this study, phytochemical composition ofArnebia densiflora(AD) was determined and cytotoxic effects of then-hexane extract and compounds isolated from this species on various cell lines were investigated. By means of serial chromatographic studies, 6 naphthoquinone derivatives were yielded, which are isovalerylalkannin,alpha-methyl-n-butyl alkannin, acetylalkannin,beta-acetoxy isovalerylalkannin, alkannin and a new compound: 4-hydroxy 4-methyl valeryl alkannin. Structures of the isolated compounds were elucidated using UV, IR, 1D-2D NMR, MS and CD methods. Cytotoxic effects of the extract and isolated alkannins were investigated on L929, HeLa, HEp-2 cells. AD and the isolated compounds demonstrated moderate to strong cytotoxic effects (IC(50)range: 4.92-172.35 mu g/ml). The results of DNA fragmentation and caspase-3 activity studies on HeLa cells exhibited that AD and the naphthoquinones isolated from it caused cytotoxicity through induction of apoptosis.