A new ergostane, named 3,16,20,22,23,25-hexahydroxyergostane, was isolated by means of chromatographic techniques from Monascus purpureus Went., which is the main component of xuezhikang capsule, a Chinese traditional patent medicine. In China, it is used to treat hyperlipidemia, atherosclerosis, cancer, and osteoporosis [1]. Because this leavening agents showed good bioactivities, a remarkable anti-hyperlipidemia drug, lovastatin, was obtained from it [2]. Previous phytochemical studies of M. purpureus led to the isolation of polyketides, monacolins, terpenes, etc. [3–6]. At current, its analog is still widely studied in the world. In our study of the chemical constituents of this leavening, its chloroform extract was investigated. A new ergostane, 3,16,20,22,23,25-hexahydroxyergostane (1), was isolated, and its structure was elucidated on the basis of spectroscopic methods. The powders of Monascus purpureus Went. (2.0 kg) were exhaustively extracted ultrasonically with petroleum ether, chloroform, and methanol at room temperature. The chloroform-soluble fraction (250 g) was chromatographed on a column of silica gel eluting with CHCl3–MeOH (gradientwise) to give four fractions (Fr.1–Fr.4). Fraction 3 (CHCl3–MeOH 90:10) was rechromatographed on silica gel eluting with CHCl3–MeOH (gradientwise) to afford a mixture containing compound 1, then the pure compound 1 (35 mg) was obtained by preparative HPLC. 3,16,20,22,23,25-Hexahydroxyergostane (1) was obtained as a colorless crystals. The molecular formula of 1 was determined to be C28H46O6 on the basis of the negative HR-ESI-MS (m/z 479.3396 [M + H] +, calcd 479.3373 for C28H47O6). The IR spectrum showed the presence of hydroxy (3531cm–1) and double-bond (1636 cm–1) groups. The 1H NMR spectrum (Table 1) of 1 in CDCl3 showed six methyls at 1.12 (3H, s, Me-18), 0.96 (3H, s, Me-19), 1.43 (3H, s, Me-21) 1.20 (3H, s, MeO-26), 1.23 (3H, s, MeO-27), and 0.85 (3H, d, J = 7.0 Hz, MeO-28), four O-bearing methines at 3.63 (1H, m, H-3), 4.66 (1H, m, H-16), 4.19 (1H, m, H-22), and 3.66 (1H, m, H-23), and two olefinic H-atom signals at 5.56 (1H, d, J = 5.5, H-6) and 5.39 (1H, d, J = 5.5, H-7). In addition, overlapped hydrogen signals at high field were observed. The above data suggest that compound 1 is similar to ergosterol [7, 8], the differences between them being the side chain and the D-ring, in which one of H-C(16) was replaced by a hydroxyl group. This is also confirmed by the 13C NMR spectrum (Table 1), in which the chemical shift of the carbon signal at position 16 takes place at 23.4 of ergosterol to 83.6 of compound 1. The 13C NMR spectrum showed 28 carbon signals, including four C=C bond C-atoms ( 140.4, 119.4, 117.1, and 138.9) and six O-bearing C-atoms (84.3, 83.6, 80.3, 74.1, 72.4, and 70.4), etc. DEPT experiments differentiated them to be 6 CH3, 6 CH2, 10 CH, 6 C. The linkage positions of substituted groups were deduced by HMQC and HMBC experiments. In the HMBC spectrum (Fig. 1), correlations from H 1.91 (H-17) to C 84.3 (C-22), from 1.43 (Me-21) to 66.8 (C-17), and from 1.12 (Me-18) to 66.8 (C-17) indicated that the side chain was attached at the C-17 position. Furthermore, correlations from H 3.66 (H-23) to C 80.3 (C-20), 74.1 (C-25), and 13.9 (C-28) and from 1.75 (H-24) to 30.2 (C-26) and 23.9 (C-27) showed their arrangement. In addition, correlations from H 5.56 (H-6) to C 40.8 (C-4) and 37.2 (C-10) and from 5.39 (H-7) to 45.9 (C-9) and 53.9 (C-14) verify the position of C=C. The correlations from H 4.66 (H-16) to C 42.9 (C-13) and from 1.87 (H-14) to 83.6 (C-16) clarified that the linkage position of the hydroxyl group of D ring was C-16. Combining all the information above, we determined the structure of 1 as 3,16,20,22,23,25-hexahydroxyergostane (1). Subsequently, the cytotoxic activity of compound 1 was evaluated against HCT-8, A549, Bel-7402, BGC823, and A2780 using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The results showed that the compound was inactive (IC50 > 10 M).