A new C-27-steroidal glycoside with three known compounds were isolated from the tuber of Ophiopogon japonicus (Thunb.) Ker-Gawl. Based on the spectral analysis, including HRMS, H-1 NMR, C-13 NMR, DEPT, H-1-H-1 COSY, HMQC and HMBC, their chemical structures were determined as pennogenin-3-O-alpha-L-rhamnopyranosyl-(1 -> 2)-beta-D-xylopyranosyl-(1 -> 4)-beta-D-glucopyranoside (1), pennogenin-3-O-alpha-L-rhamnopyranosyl-(1 -> 2)-beta-D-glucopyranoside (2), ophiopojaponin C (3) and ophiopogonin D (4). (C) 2007 Jian Zhong Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.