Spiroseoflosterol (<b>1</b>), a 7(8→9)-<i>abeo</i>-ergostane steroid with a unique spiro[4.5]decan-6-one system, was isolated from the fruiting bodies of <i>Butyriboletus roseoflavus</i>. Its structure was determined by interpretation of comprehensive spectroscopic, X-ray, and computational data. A plausible biogenetic pathway for spiroseoflosterol (<b>1</b>) was postulated based on a key semipinacol rearrangement. Compound <b>1</b> was cytotoxic to HepG2 and Huh7/S (sorafenib-resistant Huh7) with IC<sub>50</sub> values of 9.1 and 6.2 μM, respectively.