Chemical studies on Ibervillea sonorae (S. Watson) Greene root led to isolation and chemical characterization of diverse cucurbitacin triterpenoid compounds such as kinoin A, B, C, and their glucosides. In previous studies, we demonstrated that kinoin A inhibits the cell proliferation on diverse cell line and induce apoptosis in HeLa cells. Therefore, the study of the isolated compounds from the extracts continued to be necessary. The objective of the present work was to isolate and chemically characterize the active compounds of the methanolic extract of the roots of I. sonorae and to evaluate their antiproliferative activity and induction of apoptosis. By chromatographic column separation and using NMR spectroscopy experiments, cucurbitacin IIb (CIIb), known as 23,24-dihydrocucurbitacin F or hemslecin B, was isolated and identified for the first time as a chemical constituent of the crude methanolic extract of this plant. The antiproliferative activity of CIIb was evaluated by MTT assay, and the apoptosis induction capacity was monitored by annexin V-FITC/propidium iodide using flow cytometry. CIIb showed a pronounced effect on the proliferation of HeLa and A549 tumor cells, with IC50 of 7.3 and 7.8 µM, respectively, but was less effective against L929 non-cancerous murine cell line. Apoptosis induction capacity of CIIb on HeLa and A549 was monitored by annexin V-FITC/propidium iodide using flow cytometry. Exposure of HeLa and A549 with CIIb (8 µM) for 24 h increased 56.9 and 52.3% respectively of the total apoptosis compared to the negative control (p < 0.005). CIIb, isolated for the first time from I. sonorae, showed antiproliferative activity against HeLa and A549 cell lines by inducing cell death by apoptosis.