Brassinosteroids (BRs) are steroid plant hormones that are essential for many plant growth and developmental processes, including cell expansion, vascular differentiation and stress responses. Up to now the inhibitory effects of BRs on cell division of mammalian cells are unknown. To determine basic anticancer structure-activity relationships of natural BRs on human cells, several normal and cancer cell lines have been used. Several of the tested BRs were found to have high cytotoxic activity. Therefore, in our next series of experiments, we tested the effects of the most promising and readily available BR analogues with interesting anticancer properties, 28-homocastasterone (1) and 24-epibrassinolide (2), on the viability, proliferation, and cycling of hormone-sensitive/insensitive (MCF-7/MDA-MB-468) breast and (LNCaP/DU-145) prostate cancer cell lines to determine whether the discovered cytotoxic activity of BRs could be, at least partially, related to brassinosteroid-nuclear receptor interactions. Both BRs inhibited cell growth in a dose-dependent manner in the cancer cell lines. Flow cytometry analysis showed that BR treatment arrested MCF-7, MDA-MB-468 and LNCaP cells in G(1) phase of the cell cycle and induced apoptosis in MDA-MB-468, LNCaP, and slightly in the DU-145 cells. Our results provide the first evidence that natural BRs can inhibit the growth, at micromolar concentrations, of several human cancer cell lines without affecting the growth of normal cells. Therefore, these plant hormones are promising leads for potential anticancer drugs.