Abstract Invasive breast cancer is considered a great challenge for treatment of cancer in women. In 2013, an estimated 232,340 new cases of invasive breast cancer were expected to be diagnosed in the U.S., along with 64,640 new cases of non-invasive breast cancer. For women under 45, breast cancer is more common in African-American women than white women, and, overall, African-American women are more likely to die of breast cancer. CD44high/CD24low cells have been associated with invasiveness of breast cancers, in particular basal-like breast cancers. In nude mice xenografted with MDA-MB-231 breast cancer cells, CD44high/CD24low cells are the side population associated with metastasis to the bones and are considered to be cancer stem cells. The side population cells are not only involved in invasiveness of breast cancer but are resistant to chemotherapeutic drugs such as mitoxantrone and doxorubicin. The effects of an alcoholic extract of the roots of Withania somnifera (WS), a traditional herbal remedy, was tested in the sorted side population, CD44high/CD24low, of MDA-MB-231 breast cancer cells. WS caused a concentration-dependent inhibition of the sorted cells, with 95, 65, and 35% inhibition of the proliferation of cells after incubation with concentrations of 100, 50, and 25 μg/ml, respectively. Two groups of nude mice (10 mice ineach group) were xenografted with luciferase transfected CD44high/CD24low cells. Group1 served as control has been dosed with 10% DMSO via drinking water for 8 weeks. Group 2 has been given WS (300 mg/kg) via drinking water for 8 weeks. This experiment is on going and will end after 8 weeks. Tumoror size will be assessed each week using an in vivo imaging system. In conclusion, on, Withania somnifera extract showed a concentration-dependent inhibition of CD44high/CD24low cells and it is anticipated that the extract will be effective in inhibition of cell proliferation in treated mice. These findings serve as preliminary data for further preclinical studies evaluating the effects of WS. Grant support: Research supported by NIH-NCI-U54-118948 at Tuskegee University and the University of Alabama at Birmingham. Citation Format: Kamel F. Khazal. In vivo and in vitro effect of Withania somnifera extract on the proliferation of side population (CD44HIGH /CD24LOW) cells sorted from MDA-MB-231 breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5553. doi:10.1158/1538-7445.AM2015-5553