Background: Paris quadrifolia L. is a medicinal plant which contains steroidal saponins. The present study reports isolation and structural identification of six pennogenyl saponins obtained from P. quadrifolia rhizomes. The four spirostan saponins were obtained from P. quadrifolia for the first time. The cytotoxic effects of the sub-fractions and six compounds isolated from the plant extract were evaluated on tumour cells. Materials and Methods: Ethanol extract from the rhizomes of P. quadrifolia were partinioned using column chromatography. The saponins were isolated from the obtained sub-fractions by isocratic RP HPLC and their structures were determined by means of 1D and 2D NMR spectroscopy and MALDI TOF MS. The cytotoxic effects of the sub-fractions and the isolated compounds were tested against human promyelocytic leukaemia cells (HL-60), human cervical adenocarcinoma cells (HeLa) and human breast cancer cells (MCF-7) using the [(3-(4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) assay. Results: Six pennogenyl saponins were isolated from P. quadrifolia rhizomes: pennogenin 3-O-β-D-glucopyranoside (1), pennogenin 3-O-α-L-rhamnopyranosyl-(1→ 4)-β-D-glucopyranoside (2), pennogenin 3-O-α-L-rhamnopyranosyl-(1→ 2)-β-D-glucopyranoside (3), pennogenin 3-O-α-L-rhamnopyranosyl-(1→ 4)-α-L-rhamnopyranosyl-(1→ 4)-β-D-glucopyranoside (4), pennogenin 3-O-α-L-rhamnopyranosyl-(1→ 4)-[α-L-rhamnopyranosyl-(1→ 2)]-β-D-glucopyranoside (5), pennogenin 3-O-α-L-rhamnopyranosyl-(1→ 4)-α-L-rhamnopyranosyl-(1→ 4)-[α-L-rhamnopyranosyl-(1→ 2)]-β-D-glucopyranoside (6). Pennogenyl saponins 5 and 6 exhibited cytotoxic activity against HL-60, HeLa and MCF-7 tumour cells with IC 50 values of 1.0 ± 0.04 μg/ml, 1.8 ± 0.072 μg/ml and 2.4 ± 0.096 μg/ml respectively, and 2.0 ± 0.08 μg/ml, 2.5 ± 0.125 μg/ml and 3.2 ± 0.128 μg/ml respectively. Conclusion: Compounds 1 -4 were isolated from this species for the first time.