One new compound, 3,7,11,19,23,27-hexamethyl-15-hydroxymethylene-n-octacos-5,8,20-triene-10β,18α- diol-10β-D-glucopyranoside (1), along with the three known compounds, 3-epibetulinic acid (2), n-pentadecanyl octa-dec-19-en-oate (3) and β-sitosterol (4) were isolated from the methanolic extract of the cultured Catharanthus roseus hairy roots. The structures of the one new and three known compounds were elucidated using one- and two-dimensional NMR in combination with IR, EI/MS, FAB/MS. To the best of our knowledge, 3,7,11,19,23,27-hexamethyl-15-hydroxymethylene-n-octacos-5,8,20-triene-10β,18α-diol-10β-D-glucopyranoside, 3-epibetulinic acid and n-pentadecanyl octa-dec-19-en-oate were identified for the first time from the hairy roots of C. roseus.