We explored new cytotoxic C(19)-diterpenoid alkaloid, lipojesaconitine (1), from rhizoma of Aconitum japonicum THUNB. subsp. subcuneatum (NAKAI) KADOTA. Two additional non-cytotoxic new C(19)-diterpenoid alkaloids, 10-hydroxychasmanine (2) and 3-hydroxykaracoline (3), together with eight known C(19)- and C(20)-diterpenoid alkaloids (4-11) were also isolated. Their structures were elucidated by extensive spectroscopic methods including NMR (1D and 2D), IR, and MS (HRMS). Six known diterpenoid alkaloids, foresticine (5), neolinine (6), aconicarchamine A (7), 9-hydroxynominine (8), kobusine (9), and torokonine (10), were isolated for the first time from A. japonicum subsp. subcuneatum. Alkaloid 1 showed cytotoxicity with IC(50) values ranging from 6.0 to 7.3 microM against four human tumor cell lines, except a multidrug-resistant subline, suggesting that 1 was likely exported by P-glycoprotein.