A series of oxygenated yohimbane alkaloids, including three new compounds, ophiorrhines H-J (1-3), and seven known compounds, were isolated from the aerial parts of Ophiorrhiza japonica. The structures with absolute configurations were elucidated by extensive MS and NMR spectroscopic methods, as well as the single crystal X-ray diffraction and ECD calculations. Ophiorrhines H (1) and I (2) represent key oxygenated intermediates in the formation of aromatic ring E in the demethoxycarbonyl-3,14-dihydrogambirtannine (10). Ophiorrhine J (3) is a highly oxidized yohimbane derivative with the planar superconjugated system. The cytotoxic activities of all alkaloids against five human cancer cell lines were evaluated. CI - Copyright (c) 2023 Elsevier B.V. All rights reserved.