The identification and understanding of the molecular network of cancer stem cells (CSCs) have had a profound impact on our view of carcinogenesis and treatment strategy. Unfortunately, a major problem is that serial passages of CSCs from clinical solid tumor specimens currently are not available in any lab, and thus, reported data are difficult to confirm and intensively interrogated. Here, we have generated two tumor tissue-derived breast CSC (BCSC) lines that showed prolonged maintenance over 20 serial passages in vitro, while retaining their tumor-initiating biological properties. We then deciphered the intrinsic mechanism using analyses of mRNA expression array profiles. It has been determined that pro-opiomelanocortin (POMC) is closely related with protein phosphorylation mediated by G-protein-coupled estrogen receptor (GPER) in BCSC. Following, knockdown of POMC inhibits properties of mammosphere formation, CD44(+)CD24(-) population, CD44 expression, and clonogenicity ability in BCSC. We found that inhibition of POMC attenuates phosphorylation of AKT2 and GSK3 in BCSC. Further in vivo investigations demonstrated that POMC interference regulates proliferation of BCSC-bearing tumors. Combination of the clinical results that POMC positive expression is frequently upregulated in human breast cancer and POMC positivity correlated with a poor prognosis, POMC is a potential therapeutic target for BCSC. In conclusion, we have successfully established two long-term-cultured BCSC from clinical specimens. We further indicated that POMC acts as a potential therapeutic target and prognostic marker for future treatment of BCSC. What's new? Cancer stem cells (CSCs), which serve functions in tumor development and drug resistance, are of tremendous value in cancer research. Their long-term culture, however, has proven difficult. Here, two tumor tissue-derived breast cancer stem cell (BCSC) lines were successfully established and maintained in culture for more than 20 serial passages, without loss of original properties. Long-term culture enabled detailed molecular investigation, with mRNA expression analyses revealing a role for pro-opiomelanocortin (POMC) in BCSC. Knockdown of POMC attenuated tumor-initiating properties and AKT2/GSK3 phosphorylation in cells and, in vivo, inhibited BCSC proliferation. POMC was linked to poor prognosis in breast cancer patients.