Continuing work on antileukemic alkaloids of Catharanthus roseus led to the isolation of vincarodine from the post-VLB-pre-VCR fraction via pH gradient fractionation. Physicochemical analyses (IR, NMR, mass spectrometry) and chemical reactions (acetylation, reduction) were used to elucidate its structure. High-resolution mass spectrometry determined the molecular formula as C22H26N2O5 (molecular weight 398), correcting a previous dimeric proposal. Structural studies revealed an Eburna-type skeleton with a novel ether linkage, a hydroxy group at C-14, and a trans (diaxial) relationship between the oxygen atoms at C-14 and C-15. Mass spectral fragmentation patterns (e.g., base peak at m/e 200) and NMR data (including solvent shift and acetylation experiments) supported the assignment of structure 9 or its mirror image.