Breast cancer is the most common form of malignancy and the second leading cause of cancer mortality in women. The most recent epidemiological studies have shown an inverse relationship between blood lutein concentration and breast cancer risk. However, it is currently unclear whether it is lutein itself, or a metabolite of lutein that plays a role in breast tissue protection. To better understand the impact of dietary lutein on breast development, we performed an in vivo feeding study by using a modified AIN-93G diet, in which lutein was added to reach the concentration 0.01%. C57/BL6 virgin female mice at 3-week of age were fed with diets with or without lutein supplementation. Plasma, mammary gland, and liver were collected after 5 weeks, 10 weeks, and 15 weeks of feeding. Plasma lutein levels were determined by high-performance liquid chromatography (HPLC). Whole mount staining, light microscopy, and immunohistochemistry (IHC) analyses were conducted to evaluate the development of mammary glands. Our results showed that plasma lutein concentration was close to human plasma lutein level, and lutein accumulated in mammary glands in the lutein supplemented group. In the control group no lutein was detected in plasma, nor mammary gland. Our data also showed that there was no difference with regards to branching of the glands, adipocyte area, or epithelial cell proliferation between the two dietary groups. In addition, to investigate the direct effects of lutein on breast cancer cellular growth, we performed an in vitro study by using multiple human breast cancer cell lines (T47D, SKBR3, ZR-751, MCF7, MDA-MB-231). The cells were treated with lutein or beta-carotene at the concentrations ranging from 0.1 microM to 10 microM, followed by assessing one measurement of cell proliferation and the uptake of carotenoid by the cells. Our in vitro studies showed that there was no significant effect of lutein on breast cancer cell growth with each concentrations we tested. beta-Carotene, used as a positive control, led to around 30% decrease in cell growth at 1.0 microM and 10 microM. All five human breast cancer cell lines accumulated lutein to a greater extent than beta-carotene. Taken together, our data indicate that there is no effect of lutein on normal mouse breast development or human cancer cell growth. The association between plasma lutein and breast cancer risk described in previous studies might result from lutein being a biomarker of vegetable intake, or a marker of other phytochemical intake, and not a direct effect of lutein itself.