Breast cancers contain a heterogeneous population of cells with a small percentage that possess properties similar to those found in stem cells. One of the widely accepted markers of breast cancer stem cells (BCSCs) is the cell surface marker CD44. As a glycoprotein, CD44 is involved in many cellular processes including cell adhesion, migration and proliferation, making it pro-oncogenic by nature. CD44 expression is highly up-regulated in BCSCs, and has been implicated in tumorigenesis and metastasis. However, the genetic mechanism that leads to a high level of CD44 expression in breast cancer cells and BCSCs is not well understood. Here, we identify a novel cis-element of the CD44 directs gene expression in breast cancer cells in a cell type specific manner. We have further identified key trans-acting factor binding sites and nuclear factors AP-1 and NFκB that are involved in the regulation of cell-specific CD44 expression. These findings provide new insight into the complex regulatory mechanism of CD44 expression, which may help identify more effective therapeutic targets against the breast cancer stem cells and metastatic tumors.