Ten novel isocoumarins, including four pairs of enantiomers, were isolated from Artemisia dubia var. subdigitata (Asteraceae). Com-pounds 1, 2 and 3a/3b possessed a unique 6/6/6-tricyclic system comprising an unusual 1-(2-methylcyclohexyl) propan-1-one moiety fused with isocoumarin core skeleton. Compounds 4a/4b were characterized as an unexpected 2,5-dimethylcyclohexan-1-one scaffold, and compounds 5a/5b and 6a/6b were rare 1,2-seco-isocoumarin. Their structures and absolute configurations were elucidated through spectroscopic data, X-ray crystallography, ECD and NMR calculations with DP4+ analyses. Plausible biosynthetic pathways were proposed from the naturally occurring isocoumarin. Network pharmacological analysis suggested that the targets of compound 1 were significantly enriched in the cell cycle and PI3K-Akt signaling pathway. The molecular docking revealed that compound 1 had high binding affinity with CDK2 (total score: 6.8717). Furthermore, compounds 1 and 2 exhibited inhibitory activity on three human hepa-toma cell lines, with inhibitory ratios of 85.1% and 84.5% (HepG2), 88.2% and 87.3% (Huh7), and 69.2% and 69.1% (SK-Hep-1) at 200 μM, respectively.