Three new (1-3), together with three known (4-6) isoquinoline alkaloids were isolated from the whole plants of Thalictrum glandulosissimum. Their structures were elucidated by spectroscopic methods, including extensive H-1, C-13, and 2D-NMR techniques. Compounds 1-6 were evaluated for their anti-tobacco mosaic virus (Anti-TMV) activity. The results revealed that compounds 2, 3 and 4 showed potential anti-TMV activities with inhibition rates of (35.6 +/- 3.5)%, (36.2%+/- 3.8)%, (46.3%+/- 3.2)%, at the concentration of 20 mu M, respectively. These rates are higher than that of positive control.