A phytochemical investigation on the roots of Aconitum austroyunnanense led to the isolation of two previously undescribed diterpenoid alkaloids, austroyunnanines D and E (1 and 2). Their structures were determined by spectral methods such as 1D and 2D (H-1-H-1 COSY, HMQC, NOESY, and HMBC) NMR spectroscopy, in addition to high-resolution mass spectrometry. The isolated compounds were tested in vitro for cytotoxic activity against three human gastric carcinoma cell lines. As a result, compound 1 exhibited some cytotoxicities against all the tested tumor cell lines with IC50 values less than 20.0 mu M.