A phytochemical work on the alkaloid constituents from Trigonostemon lii Y.T. Chang was conducted to give six new β-carboline alkaloids, trigonostemines A–F (1–6) and eight known β-carboline alkaloids (7–14). Their structures were elucidated by extensive spectroscopic techniques including 2D NMR experiments and mass spectrometry. All of the compounds were evaluated for their cytotoxic activities against the HL-60, SMMC-7721, A-549, MCF-7, and SW480 human cancer cell lines. Trigonostemines A and B (1 and 2) exhibited stronger inhibitory activities than the positive control (cisplatin) in some cell lines.