Silibinin, with a strong antioxidant activity and a weak cytotoxic property, is considered a candidate for cancer prevention. As there is no information on its effect on breast cancer tumor-initiating cells [cancer stem cells (CSCs)] in a 3D culture model, which more closely mimic natural tissues, we carried out this study to determine whether silibinin can target breast CSCs in MDA-MB-468 cells cultured under 3D and 2D conditions. Silibinin was added to culture medium of MDA-MB-468 at a half maximal inhibitory concentration (IC50) dose in 2D and 3D models. Then, stemness properties were assessed using colony and sphere-formation tests. Flow cytometry and real-time PCR were used to determine the different expression levels of stem cell-related marker at protein and mRNA levels under both culture conditions. Our results showed that silibinin inhibits cell growth in a dose-dependent manner by induction of apoptosis, alteration of the cell cycle, reduction of stemness properties and function, and induction of tumoral differentiation. The mechanism of silibinin action and also the response of tumor cells differed when cells were cultured in a 3D model compared with a 2D model. Silibinin may potentially target breast CSCs. Moreover, tumor-initiating cells are more sensitive to silibinin in a 3D culture than in a 2D culture.