The present study investigated the influence of anti-estrogen treatment (fulvestrant) on pituitary adenoma cell line GH3 biological activity, the estrogen receptor alpha pathway, the WnT pathway, and mechanisms of decreased Wnt inhibitory factor-1 expression in GH3 cells. Results showed that fulvestrant suppressed GH3 cell proliferation and reduced hormone secretion in a dose-dependent manner. Estrogen receptor alpha and Wnt4 expression decreased, but Wnt inhibitory factor-1 expression increased in a dose-dependent manner following fulvestrant treatment, and beta-catenin expression remained unchanged. Inhibitors of DNA methylation and histone modification upregulated Wnt inhibitory factor-1 expression. Results suggested that fulvestrant suppressed biological activity of GH3 cells via the estrogen receptor alpha and Wnt pathways. These results suggested that decreased Wnt inhibitory factor-1 expression in GH3 cells played a role in epigenetic mechanisms. Anti-estrogen therapies could provide novel treatments for growth hormone adenomas.