A new withanolide, dinoxin B (12,21-dihydroxy-1-oxowitha-2,5,24-trienolide-27-O-β-D-glucopyranoside, 1), was isolated from a methanol extract of Datura inoxia leaves, using bioassay-guided fractionation. The structure was determined by spectroscopic techniques, including (1)H, (13)C, and 2D NMR experiments as well as by HRMS. Extracts and the purified compound were tested for their antiproliferative activities toward a panel of human normal and cancer cell lines. Dinoxin B (1) and its aglycone (2) exhibited submicromolar IC(50) values against multiple human cancer cell lines. Among the most sensitive were several breast cancer cell lines. Dinoxin B (1) was found only in D. inoxia and was not detected in D. metel or D. stramonium. The accumulation of this compound was limited largely to leaf tissue, with little to none detected in extracts from the flowers, fruits, roots, or stems of D. inoxia.