Cucumis bisexualis has been widely served as a beneficial wild fruit in China. Four new homoisoflavonoids (1-4), together with eight known homoisoflavonoid derivatives (5-12) were isolated from the 90% EtOH extract of C. bisexualis, using repeated open column chromatography and preparative thin-layer chromatography. They were elucidated based on the analysis of spectral data and references from the fruits of C. bisexualis for the first time. These compounds (1-12) were evaluated for their in vitro inhibitory effects against the increase of aspartate aminotransferase and alanine aminotransferase levels in H O -induced HepG2 cells in the range of their maximum nontoxic concentration. Among them, compounds 3, 4, 8, and 9 exhibited certain hepatoprotective activities. PRACTICAL APPLICATIONS: Cucumis bisexualis A.M. Lu ＆ G.C. Wang is an edible and medical fruit with many functional properties. A detailed study was made to identify the bioactive constituents of C. bisexualis and four new homoisoflavonoids (1-4) and eight known homoisoflavonoid derivatives (5-12) were isolated from the fruits of C. bisexualis for the first time. Compounds 3, 4, 8, and 9 exhibited certain hepatoprotective activities. This study suggests that natural compounds isolated from C. bisexualis could be used as promising hepatoprotective agents.