A novel gamma-methylidene-spirobutanolide spirolephtoshol (1) was isolated from ascomycetous fungus Leptosphaeria doliolum as a cytotoxic compound. The relative structure was established by the NMR analysis involving the NOE experiments. Absolute structure of the bicyclic moiety was determined by chemical derivation followed by the CD analysis. The relative and absolute stereochemistry of the side chain was established by comparison of the (1)H NMR spectra and the chiral GC chromatograms of the degradation product with the synthetic samples.