A new prenylated indole alkaloid—Penicimutamide CN-oxide (1), a new alkaloid penicimutamine A (2), along with six known alkaloids were isolated from an endophytic fungusPallidocercospora crystallina. A simple and accurate method was used to determine the N-O bond in theN-oxide group of1. By using a β-cell ablation diabetic zebrafish model, compounds1,3,5,6and8showed significantly hypoglycemic activities under the concentration of 10 μM. Further studies revealed that compounds1and8lowered the glucose level through promoting glucose uptake in zebrafish. In addition, all eight compounds showed no acute toxicity, teratogenicity, nor vascular toxicity in zebrafish under the concentrations range from 2.5 μΜ to 40 μM. Importantly, these results provide new lead compounds for the development of antidiabetes strategies.