Visceral hypersensitivity (VH) is common in irritable bowel syndrome (IBS), and female patients are more likely to seek health care services for IBS-related abdominal pain. Estrogen has been reported to mediate pain modulation via its receptor, and mast cells are known to participate in the development of visceral hypersensitivity. Our previous studies showed that the G-protein-coupled estrogen receptor (GPER, also known as GPR30) was expressed by mast cells in human colonic tissues and was associated with IBS type and severity of visceral pain. However, whether GPER is involved in estrogen-dependent visceral hypersensitivity via mast cell degranulation is still unknown. Rats were subjected to wrap partial restraint stress to induce visceral hypersensitivity, and were ovariectomized (OVX) to eliminate the effects of estrogen on visceral hypersensitivity. OVX rats were treated with estrogen, an estrogen receptor α and β antagonist (ICI 182.780), a GPER antagonist (G15) or a GPER agonist (G1), to evaluate the effects of estrogen via its receptor. The colorectal distention test was performed to assess visceral sensitivity. Immunofluorescence studies were performed to evaluate GPER and mast cell tryptase co-expression. Mast cell number with degranulation was detected by specific staining. Mast cell tryptase expression in rat colon was also investigated by Western blot and immunohistochemistry. Substance P and histamine expression were examined by ELISA. GPER was expressed by the majority of tryptase-positive mast cells in the colonic mucosa. Stressed rats showed increased visceral sensitivity, increased mast cell degranulation, mast cell tryptase expression and increased colon histamine levels. Ovariectomy reduced stress-induced VH in female rats and decreased mast cell degranulation, mast cell tryptase expression and histamine levels, whereas estrogen replacement reversed these effects. In OVX rats, the GPER antagonist G15 counteracted the enhancing effects of estrogen on stress-induced VH, mast cell degranulation, mast cell tryptase and histamine expression, whereas VH was preserved after treatment with ICI 182.780. On the other hand, pretreatment with the selective GPER agonist G1 at doses between 1 and 20 μg/kg significantly increased VH, mast cell tryptase and histamine expression in OVX stressed rats, mimicking the effects of estrogen. GPER plays a pivotal role in the regulation of mast cell degranulation, mast cell tryptase expression and histamine levels and contributes to the development of colonic hypersensitivity in a female rat model of IBS.© 2020 Société Française de Pharmacologie et de Thérapeutique.