Breast cancer and obesity are serious health problems and their relationship has been studied for many years. Leptin is mainly secreted by adipocytes and plays a key role in breast cancer development. Leptin expression is up-regulated in obese individuals and promotes breast cancer cell growth. On the other hand, exposure to environmental estrogens has been found to be directly related to breast cancer. Zeranol (Z) is a non-steroidal anabolic growth promoter used in the beef industry in the US. This study focused on the evaluation of Z and Z-containing sera (ZS) and its adverse health risk to human consumption of Z-containing meat produced from Z-implanted beef cattle. We hypothesized that Z increases the risk of breast neoplasia in women, particularly in obese women. A cell proliferation assay, ELISA analysis, RT-PCR and Western blot analysis were conducted. Our study demonstrated that Z and ZS collected from Z-implanted heifers stimulated the proliferation of primary cultured human normal breast epithelial cells (HNBECs) by up-regulating cyclin D1 expression. Leptin increased the sensitivity of HNBECs to Z, and Z increased the ability of HNBECs to secrete leptin. These results suggest an interaction between leptin and Z in HNBECs. Furthermore, Z may play a role in leptin-induced breast neoplasia.