AbstractPhytochemical study on the root tubers of Ophiopogon japonicus resulted in the isolation and identification of 13 homoisoflavonoids, including three new compounds, 8‐formyl‐7‐hydroxy‐5,4′‐dimethoxy‐6‐methylhomoisoflavone (1), 6‐formylisoophiopogonone B (2), and 8‐formylophiopogonanone B (4), and the ten known homoisoflavonoids 3, and 5–13. The absolute configurations of 8‐formylophiopogonanone B (4) and 8‐formyl‐7‐hydroxy‐5,4′‐dimethoxy‐6‐methylhomoisoflavanone (5) were confirmed by time‐dependent density‐functional‐theory (TD DFT) calculations of their theoretical electronic circular dichroism (ECD) spectra. The structure of the formerly reported ‘6‐aldehydoisoophiopogonone B’ was revised to 8‐formylophiopogonone B (3). All compounds were evaluated for their cytotoxic activities against the human‐lung‐tumor A549 cell line, and compounds 3, 9, 10, and 13 exhibited promising antiproliferative activities with IC50 values of 10.01, 6.40, 0.84, and 1.66 μM, respectively.