Seven previously undescribed diterpenoid alkaloids, including five C20-diterpenoid alkaloids, barpuberudine, barpubesines A-D, and two C18-diterpenoid alkaloids, barpubenines A-B, along with 11 known diterpenoid alkaloids were isolated from the whole plant of Aconitum barbatum var. puberulum Ledeb. (Ranunculaceae). Barpuberudine is an unprecedented carbon skeleton of C20-diterpenoid alkaloid, while barpubenines A-B are the first example of rearranged types in C18-diterpenoid alkaloids. Their structures were elucidated based on a comprehensive spectroscopic data analysis. The probable pathway of biogenesis of barpuberudine and barpubenines A-B were discussed. Additionally, the antiarrhythmic, cytotoxic and antimicrobial activities of isolates were also evaluated.