BACKGROUND: Non-functioning pituitary adenomas (NFPAs) are common pituitary tumors, and surgery is generally the only treatment option. Few attempts have been made to explore target molecules for the development of NFPA pharmacological treatments. METHOD: We quantitatively assessed the expression profiles of estrogen receptor (ER) transcripts and proteins in NFPA samples, using reverse transcription-digital polymerase chain reaction (RT-dPCR) and immunohistochemistry, and further investigated the correlations between the expression levels of ER and those of downstream responsive genes. All patients had undergone surgery at the same high-volume hospital. A total of 20 patients with NFPAs were included. All patients were new-onset, and none were diagnosed with intratumoral hemorrhages or cysts. RESULTS: NFPA samples exhibited a bimodal ESR1 expression pattern and were categorized into significantly different high- and low-ESR1 expression level groups (P < 0.05). In contrast, expression levels of ESR1 variants and ESR2 could barely be detected. Similar results were obtained through the immunohistochemical staining of NFPAs, using well-validated antibodies against ERs. The expression levels of ESR1 positively correlated with those of GREB1, an estrogen-responsive gene [correlation coefficient (r) = 0.623, P = 0.003]. CONCLUSIONS: ESR1 expression levels in NFPAs exhibited a bimodal pattern and were positively correlated with GREB1 expression levels. The accurate assessment of ER expression levels may further advance future NFPA-related research.