A new coprostanol glycoside, 26-trihydroxy-16, 22-dioxo-3β-[(α-Lrhamnopyranosyl)oxy]-5β-cholestan-1β-yl O-α-L-rhamnopyranosyl-(1→2)-β-Dfucopyranoside (1) (25S)-5β-spirostan-1β,2β,3β,4β,5β,6β-hexol (2), a new C-22 steroidal lactone saponin, (20R)-16β-trihydroxy-3β-[(α-L-rhamnopyranosyl)oxy]-1β-[(O-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyl)oxy]-5β-pregnane-20-oic acid ᵧ-lactone (3) along with two known pregnane glycosides (4 and 5) were obtained from the roots of Reineckia carnea. Their structures were determined by 1D, 2D NMR, IR and MS data analysis. In addition, the cytotoxic activities in HT-29, HCT116, H1299 and A549 tumor cells of the isolated compounds (1 − 5) were determined by the MTT method. The results showed that only compound 1 exhibited weak effect against these cells with IC 50 values ranging from 63.36 μM to 105.01 μM.