The aim of the present study was to investigate the effect of anti-estrogen treatment (fulvestrant) on the biological activity of hepatocellular carcinoma (HCC), involving the estrogen receptor α (ERα) and Wnt pathways, and to evaluate whether ERα and Wnt inhibitory factor-1 (WIF1) could be biomarkers for anti-estrogen clinical therapy. H22 and HepG2 cells were treated with 0.04 to 625 nM fulvestrant and the WST-8 method was used to assess the inhibition rate after 72 h. Furthermore, prolactin (PRL) secretion by HepG2 cells was assessed at 24 h using an enzyme immunoassay. Quantitative polymerase chain reaction and western blot analysis were used to analyze the mRNA and protein expression levels of ERα, β-catenin and WIF1, respectively, in HepG2 cells. For clinical patient analysis, the tumor volume was analyzed by magnetic resonance imaging methods, and PRL in the blood was detected by an enzyme immunoassay. In HepG2 cells, the mRNA and protein expression levels of ERα were downregulated (P<0.01), while β-catenin expression remained unchanged and WIF1 expression was upregulated (P<0.01). Analysis of samples from clinical patients demonstrated that there was a positive correlation between PRL levels and tumor volume. In addition, as compared with non-cancerous tissues, the ERα mRNA levels in tumor tissue were upregulated (P<0.05), particularly in that of male patients, while WIF1 expression was significantly downregulated (P<0.01). In conclusion, fulvestrant inhibited the proliferation of HepG2 cells, involving the ERα and non-canonical Wnt pathways, and it may be a promising therapeutic for HCC.