Abstract: To obtain a structurally novel steroid-derived chiral hydroxyl-containing polyene rigid spirocycle and to explore its antitumor activity and underlying mechanism, the target compound was synthesized from androstenedione dimer. Under nitrogen protection at 0–5 ℃, sodium borohydride （molar ratio to substrate 4.2） was uniformly added to an ethanol solution of the dimer over 2.0 h for regioselective reduction, followed by dilute hydrochloric acid-catalyzed elimination. The structure was confirmed by LC-MS, NMR spectroscopy, and single-crystal X-ray diffraction, and the in vitro antitumor activity and mechanism were evaluated through cell-based assays. The results showed that under the optimized conditions, the yield reached 73.0%. The spiro center and the two chiral hydroxyl-bearing carbon atoms on the five-membered rings were all assigned as S configuration. The compound exhibited significant antiproliferative activity against colorectal cancer HCT-116, breast cancer MCF-7, cervical cancer HeLa, and hepatocellular carcinoma HepG2 cells, with half-maximal inhibitory concentration （IC₅₀） values of 3.19 ± 0.35, 6.92 ± 0.68, 10.41 ± 1.90, and 11.67 ± 1.49 μM, respectively. Mechanistic studies revealed that it induced intracellular reactive oxygen species （ROS） accumulation in HCT-116 cells in a concentration-dependent manner. These findings indicate that this steroid-derived spirocycle may exert its antitumor effects via the oxidative stress pathway, providing a reference for the design of novel steroidal antitumor drugs based on ROS modulation.