Rosellinia necatrix is a pathogenic fungus which causes white root rot to potato, soybean, tea, peanut, apple and so on and produces cytochalasin E with physiological activity towards mammalian cells, microorganisms and plants. We searched in detail for new plant growth regulators in the metabolites of this fungus and succeeded in isolating the compound designated rosellichalasin (1) together with cytochalasin E (2). This paper deals with the isolation and structural elucidation of rosellichalasin. The fungus was statically cultured in a malt extract medium containing 3% peptone at 24°C for 21 days. Dry mycelial mats were extracted with acetone, and the extract was purified by silica gel column chromatography (benzene-acetone) and Sephadex LH-20 column chromatography (methanol) to obtain colorless needles of 1 (78 mg, mp 121~123°C, [α]D²⁰ -2.7°) and 2 (1.48 g). The molecular formula of 1 was determined to be C28H33NO5 by HRMS. IR (1665, 1640, 1718 cm⁻¹), UV (245 nm, ε=12500), EIMS (m/z 463, fragment ions m/z 372, 190, 120, 91) and ¹H/¹³C-NMR data (comparison with 2) indicated 1 was a cytochalasin analog. A comparison of ¹³C-NMR spectral data between 1 and 2 showed the carbonate carbon at δC 149.3 (C-21) in 2 was shifted to δC 171.8 (ester carbon) in 1, and structural analysis revealed a trans α,β-unsaturated ketone system (-CH2-CH=C(CH3)-) in 1. On the basis of these results, the structure of 1 was established as 6,7-epoxy-10-phenyl-5,6,16,18-tetramethyl-22-oxa-[12]-cyclochalas-13,19-diene-17,21-dione. In a bioassay using lettuce seeds, 1 at a dose of 100 mg/l inhibited germination by 50% after 24 hr, and root and hypocotyl growth by 70% and 60% at the same concentration, respectively.