Two new tricyclic alkaloids (1 and 2), together with three known (3–5) alkaloids, were isolated from the whole plants of Thalictrum cultratum. Their structures were determined by means of HR-ESI-MS and extensive 1D and 2D NMR spectroscopic studies. Compounds 1–5 were tested for their anti-rotavirus activity. The results revealed that compounds 1–5 exhibited potent anti-rotavirus activity with therapeutic index (TI) values in the range 10.7–17.7. © 2020, Springer Science+Business Media, LLC, part of Springer Nature.