MicroRNA-26a (miR-26a) is a tumor suppressor that is reduced in hepatocellular carcinoma (HCC). Increasing evidence indicates that the liver is a hormone-responsive organ like the breast. The purpose of this study was to investigate whether miR-26a, regulated by a human α-fetoprotein (hAFP) and human telomerase reverse transcriptase (hTERT) dual promoter, could be specifically expressed in liver tumor cells to suppress their growth and to clarify whether estrogen receptor-α (ERα) is regulated by miR-26a and involved in the HCC process. Our data show that miR-26a expression driven by a hAFP-TERT dual promoter was tumor-specific and decreased the viability of tumor cells by regulating ERα, progesterone receptor (PR) and P53 except for cyclin D2 or cyclin E2 in vitro and in vivo. Our data also show that estradiol (E2) promotes the growth of liver cancer cells similar to breast cancer cells partly via the E2-ERα pathway and that miR-26a significantly down regulates ERα and prevents the stimulation of hepatoma cell growth by E2. These data suggest that ERα, which is regulated by miR-26a, is important for liver tumor cell growth. Moreover, hAFP-TERT dual promoter-mediated miR-26a expression could specifically exert potential antitumor activity and provide a novel targeting approach for cancer therapy.