Veratrum dahuricum (Liliaceae), growing in the northeast of China, has rhizomes used in traditional Chinese medicine for apoplexy, epilepsy, and acariasis. Previous phytochemical studies on the genus Veratrum reported about 100 steroidal alkaloids, including 11 from V. dahuricum. Herein, we investigated steroidal alkaloids of V. dahuricum collected in Jilin Province, P. R. China. Air-dried and powdered rhizomes (25 kg) were refluxed with 75% ethanol, concentrated, partitioned with petroleum ether, chloroform, ethyl acetate, and n-butanol. The chloroform and n-butanol portions were subjected to silica gel column chromatography and other purification methods. Thirteen compounds were isolated and identified as maackinine (1), 3-acetyl-15-methylbutylgermine (2), veramarine (3), cyclopamine (4), veratramine (5), jervine (6), 3,15-diangeloylgermine (7), 3-angeloylzygadenine (8), 3-veratroylzygadenine (9), 15-angeloylgermine (10), zygadenine (11), germine (12), and veratrosine (13), among which compounds 1–4, 7, 8, 10, 11 were isolated from V. dahuricum for the first time. The potential antitumor activities of compounds 4–10 were evaluated by MTT assay with A549, LOVO, QGY-7703, and 6T-CEM cell lines. These compounds were moderately inhibitory to the four cell lines; overall, jerveratrum-type alkaloids (4–6) exhibited stronger antitumor activity than cevine alkaloids (7–10), while compounds 6 and 10 showed superior inhibitory effect on the 6T-CEM cell line with IC50 at 6.83 and 8.74 μg/mL, respectively.