A phytochemical investigation of the EtOH extract from the rhizomes and roots of Smilax scobinicaulis resulted in the isolation of a new isospirostanol-type steroidal saponin, namely (25 R)-5α-spirostan-3β,6β-diol 3-O-β-D-glucopyranosyl-(1 → 4)-[α-L-arabinopyranosyl-(1 → 6)]-β-D-glucopyranoside (1), along with four known steroidal saponins (2-5). The structures of these compounds were determined by 1D- and 2D-NMR spectroscopic analysis, FABMS and HR-ESI-MS as well as chemical degradation. The isolated saponins were evaluated for their in vitro cytotoxicity against A549, LAC and Hela human cancer cell lines, which demonstrated that only compound 1 possessed significant cytotoxic activity with IC₅₀ values of 3.70, 5.70 and 3.64 µM, respectively.