Veratrum nigrum is recognized as a medicinal plant used for the treatment of hypertension, stroke, and excessive phlegm. Chemical investigation of the roots and rhizomes led to the isolation of five new steroidal alkaloids, jervine-3-yl formate (1), veramarine-3-yl formate (2), jerv-5,11-diene-3β,13β-diol (3), (1β,3β,5β)-1,3-dihydroxyjervanin-12(13)-en-11-one (4), and veratramine-3-yl acetate (5). Compounds 1 and 5 exhibited potent inhibitory activity (11.3 and 4.7 μM, respectively) against protein tyrosine phosphatase 1B (PTP1B), which has emerged as a viable target for treatment of type 2 diabetes mellitus. On the basis of their PTP1B inhibitory activity, the compounds were evaluated for their potential to enhance glucose uptake in C2C12 skeletal muscle cells. The insulin-stimulated glucose uptake was enhanced upon treatment with compounds 1 and 5 (10 μM) by 49.9 ± 6.5% and 56.0 ± 9.7%, respectively, in a more potent manner than that with the positive control rosiglitazone (47.3 ± 3.4% at 30 μM). These results suggest that steroidal alkaloids serve as practical antidiabetes mellitus leads capable of enhancing glucose uptake.