Hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). Emerging evidence has shown the association between aberrantly expressed miR-221 and cancer development; however, little is known concerning its potential role in hepatitis B virus (HBV)-related HCC. In the present study, functional studies demonstrated that HBx leads to the promotion of cell proliferation and cell growth viability. Obviously overexpressed miR-221 was found in HBx-transfected cells compared with the mock counterparts. Suppression of miR-221 significantly inhibited HCC cell proliferation. Western blot analysis indicated that estrogen receptor-α (ERα) was downregulated in HCC tissues and cell lines. Bioinformatic analysis combined with validation experiments identified ERα as a direct target of miR-221. The present study suggests that miR-221 modulates HCC cancer cell proliferation by suppressing ERα, functioning as a tumor promoter. Moreover, our data imply that miR-221 has potential as an miRNA-based therapeutic target for HBV-related HCC.