The sea hare Aplysia kurodai Baba has been known to contain various unique metabolites. Herein we describe the isolation of a new type of potent antitumor compound termed aplyronine A (1) and the congeners aplyronines B (2) and C (3) from the same animal and report their structures. Originally, aplyronine A (1) was isolated by eight-step chromatographic separation guided by the cytotoxicity against HeLa-S3 cells in vitro. Subsequently, a more efficient method was developed for the isolation of 1, enabling isolation of the congeners aplyronines B (2) and C (3). The ethyl acetate-soluble material from the sea hare was separated by solvent partitioning and chromatography to afford aplyronine A (1) (2.5 × 10⁻⁵% yield based on wet weight), aplyronine B (2) (1.4 × 10⁻⁵% yield), and aplyronine C (3) (3 × 10⁻⁷% yield) as amorphous powders, respectively. Aplyronines A (1), B (2), and C (3) showed strong cytotoxicities against HeLa-S3 cells in vitro with IC₅₀ of 0.039, 4.39, and 159 ng/mL, respectively. Aplyronine A (1) exhibited exceedingly potent antitumor activities in vivo against P388 murine leukemia (T/C = 545%, 0.08 mg/kg), Lewis lung carcinoma (T/C = 556%, 0.04 mg/kg), Ehrlich carcinoma (T/C = 398%, 0.04 mg/kg), colon 26 carcinoma (T/C = 255%, 0.08 mg/kg), and B16 melanoma (T/C = 201%, 0.04 mg/kg). Structure determination was carried out with aplyronine A (1) in details: [α]²⁸ᴰ +32° (c 0.26, MeOH); UV (MeCN) λₘₐₓ 256 nm (ε 30 000); IR (CHCl₃) 3690, 3500, 1730, 1690, and 1655 cm⁻¹. The molecular formula of 1 was established to be C₅₉H₁₀₁N₃O₁₄ by HRFABMS [(M + H)⁺ m/z 1076.7360, Δ -0.2 mmu] and NMR data. The IR spectrum indicated the presence of hydroxyl groups, confirmed by acetylation (Ac₂O, pyridine) to give the diacetate 4. The ¹H and ¹³C NMR spectral data showed the presence of an α,β,γ,δ-unsaturated ester (E,E geometry), an acetate, two additional esters, five olefins (two corresponding to the α,β,γ,δ-unsaturated ester), three methoxy groups, and two dimethylamino groups. The UV spectrum confirmed the α,β,γ,δ-unsaturated ester group. A terminal N-methyl-N-vinylformamide structure in 1 was deduced when the ¹H NMR data were compared with those for scytophycins, sphinxolide, and macrocyclic trisoxazoles. Owing to the restricted rotation about the N-methyl-N-vinylformamide terminus (2:1 ratio) and the presence of two scalemic amino acid portions (1.1:1 and 3:1 ratios for N,N,O-trimethylserine and N,N-dimethylalanine moieties, respectively), doubled NMR signals for some protons and carbons were observed. Detailed analysis of ¹H-¹H COSY, phase-sensitive ¹³C-¹H COSY (JCH = 135 Hz), and HMBC experiments on 1 and its diacetate 4 established the connectivity of partial structures (e.g., C2-C9, C14-C17, C18-C25, C27-C34, C2'-C3', C2''-C3'') and positions of functional groups (four esters, three methoxy groups, two dimethylamino groups). The E geometry of the C14-C15 double bond was clarified by NOEs between H13 and H15 (8%) and between 14-Me and H16 (6% and 3%).