In previous papers, we reported three new natural diketopiperazines, tryprostatins A, B and demethoxyfumitremorgin C together with fumitremorgin C, 12,13 dihydroxyfumitremorgin C, fumitremorgin B and verruculogen, as a new group of M-phase inhibitors of the mammalian cell cycle, isolated from the secondary metabolites of Aspergillus fumigatus. To obtain larger amounts of those compounds for detailed study of their biological activities and mechanism of action, we carried out large scale fermentation of the producing strain. During the preparation, we isolated a novel compound named spirotryprostatin B (1) which inhibited the cell cycle progression of tsFT210 cells at the G2/M phase. This communication reports the isolation, structure and biological activities of 1. Spirotryprostatin B (1) was obtained as a slightly yellow-colored crystalline solid with melting point 137-138°C and [α]²⁵ -162.1° (c 0.92, CHCl₃). Its molecular formula was C₂₁H₂₁N₃O₃ (determined by HR-EI-MS). UV spectrum showed absorption maxima at 212 (ε 36660), 227 (sh, 28820), 242 (sh, 24830), 272 (sh, 17350) and 286 nm (sh, 14810); IR spectrum showed absorption at 3440, 3240 (N-H), 1730 (γ-lactam C=O), 1680, 1655 (amide C=O) and 1640 cm⁻¹ (C=C). Its structure, elucidated by 2D NMR techniques (PFG-HMBC, DEPT, WHCOSY, PFG-HMQC) and NOE experiments, has an unique spiro ring system composed from a γ-lactam fused to a benzene ring and a pentacyclic enamine fused to a diketopiperazine moiety, derived from a tryptophan unit, a proline residue and an isoprenyl group. Spirotryprostatin B (1) completely inhibited the cell cycle progression of tsFT210 cells in the G2/M phase at final concentrations over 12.5 μg/ml. It also showed cytotoxic activity on human chronic myelogenous leukemia K562 cells and human promyelocytic leukemia HL-60 cells with MIC values of 35 μg/ml and 10 μg/ml, respectively. The present result provides spirotryprostatin B (1), a novel natural diketopiperazine derivative having an unique spiro ring skeleton, as a new inhibitor of the mammalian cell cycle.