Estrogen is suggested to participate in pathogenesis of irritable bowel syndrome (IBS), but expression of G protein-coupled estrogen receptor (GPER) in the colon of IBS patients has never been investigated. The aim of this study was to investigate the expression of GPER and classical estrogen receptors in the colon of IBS patients and healthy controls. Colonic biopsies were obtained by endoscopy from patients with IBS (n= 46) and healthy subjects (n= 13). Expression of GPER, estrogen receptor α (ERα) and estrogen receptor β (ERβ) in mast cells were measured by double-labelling immunofluorescence. Quantification of mRNA expression was performed for GPER, ERα and ERβ by real-time polymerase chain reaction. Differential distribution of GPER, ERα and ERβ were detected in human colonic mucosa. The expression of GPER in the cytoplasm of mast cells and GPER-positive cells was significantly higher in diarrhea-predominant IBS (D-IBS) patients than that in constipation-predominant IBS (C-IBS, P< 0.001) patients and healthy subjects (P= 0.005). ERα and ERβ were not detected in majority of mast cells in colonic mucosa and no difference of immunostaining results for ERα and ERβ was found among these three groups. A positive correlation (r= 0.451, P= 0.011) between GPER-positive cell counts and abdominal pain severity was observed in D-IBS group. Relative mRNA expression of GPER in D-IBS was also higher than that in C-IBS (P= 0.018) and healthy subjects (P= 0.011). The present study, for the first time, demonstrated the expression of GPER in human colonic mucosa and its correlation with abdominal pain severity.