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Two new C27-steroidal glycosides were isolated from the fibrous roots of Ophiopogon japonicus. The spectral analysis and chemical evidence revealed their chemical structures to be (25R)-spirost-5,14-dien-3β-yl-O-α-L-rhanmopyranosyl-(1→2)-β-D-xylopyranosyl-(1→4)-β-D-glucopyranoside (1) and ophiogenin 3-O-β-D-glucopyranoside (2).