Iminimycin B, a novel indolizine alkaloid featuring a rare pyridinium, was isolated from the cultured broth of a streptomycin-producing strain, Streptomyces griseus OS-3601, through a physicochemical screening method. Its structure was elucidated on the basis of mass and NMR analyses. Stereochemical assignment of iminimycin B was archived by NMR studies, electronic circular dichroism (ECD) analysis, and advanced Marfey's method.