A phytochemical investigation on the seeds of Sophora alopecuroides led to obtaining fourteen structurally diverse matrine-based alkaloids (1—14), including eight new ones (1, 6, 8—12, 14). Notably, alopecuroide F (1) represents the first dimeric matrine-type skeleton assembled via unprecedent C-13–C-12' connection. The new structures were determined through extensive spectroscopic data (UV, OR, HRESIMS, 1D, and 2D NMR), ECD calculations, and three instances, verified by X-ray crystallography. Biologically, all alkaloids were evaluated for cytotoxicity against four human cancer cell lines (HepG2, A549, THP-1, and MCF-7) and anti-inflammatory activities for two pro-inflammatory cytokines (TNF-α and IL-6). Alopecuroide F (1) can inhibit TNF-α and IL-6 productions in a dose-dependent manner, with IC50 values of 35.6 ± 0.5 and 41.7 ± 0.8 μmol/L, respectively. © 2021 SIOC, CAS, Shanghai & WILEY-VCH GmbH