Three new steroidal alkaloids 1–3, together with four known compounds 4–7, were isolated from the ethanol extract of Veratrum grandiflorum Loes. Their structures were elucidated by NMR (1D and 2D NMR) and MS spectroscopic data. The inhibition activities on Hedgehog (Hh) pathway were evaluated using a cell-based bioassay system (Shh-LIGHT 2 cells). The results showed that compounds 1–3 and 5 displayed inhibitory activities obviously with the IC50values of 0.63–3.11 μM. Among them, compound 5 showed the most prominent inhibition activity (IC50 = 0.63 ± 0.02 μM). Thus, these active alkaloids may be potent natural compounds as Hh pathway inhibitors for the treatment of various cancers. © 2016 Elsevier Ltd