Estrogens exert immunomodulatory action in many autoimmune diseases. Accumulating evidence highlights the meaningful impact of estrogen receptors in physiology and pathophysiology of the colon. However, the significance of G protein-coupled estrogen receptor (GPER) on Crohn's disease (CD), one of the inflammatory bowel disease (IBD) types, is still elusive. Our study revealed GPER overexpression at the mRNA and protein levels in patients with CD. To evaluate the effects of GPER activation/inhibition on colitis development, a murine 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced model of CD was used. We showed that activation of GPER reduces mortality, improves macroscopic and microscopic scores and lowers C-reactive protein (CRP) level. The impact of estrogen signaling on the suppression of the intestinal inflammation was proved by immunohistochemistry. It was demonstrated that GPER activation is accompanied by modulation of extracellular-signal regulated kinase (ERK) signaling pathway and expression level of genes involved in signal transmission and immune response as well as the expression of some microRNAs (miR-145, miR-148-5p and miR-592). Our study revealed that the membrane-bound estrogen receptor GPER mediates anti-inflammatory action and seems to be a potent therapeutic target in maintaining remission in CD.