A new cytisine-type alkaloid, along with five known alkaloids was obtained from the seeds of Sophora alopecuroides. Their structures were determined to be (−)-N-(2'-hydroxy-3',5'-di-tert-butyl-toluene)-cytisine (1), (−)-lupanine (2), (+)-matrine (3), (+)-sophoramine (4), (+)-lehmannine (5) and (−)-sophocarpine (6). Their structures were established by NMR, ECD and HRESIMS data analyses. Their cytotoxicity effects against five human tumor cell lines were tested by MTT assay. Compound 1 has showed a wide range of cytotoxicity activities against varied tumor cells in vitro. © 2020 Informa UK Limited, trading as Taylor & Francis Group.