The inhibition of the G protein-coupled estrogen receptor (GPER) offers promising perspectives for the treatment of breast tumors. A peptide corresponding to part of the hinge region/AF2 domain of the human estrogen receptor α (ERα17p, residues 295-311) exerts anti-proliferative effects in various breast cancer cells including those used as triple negative breast cancer (TNBC) models. As preliminary investigations have evoked a role for the GPER in the mechanism of action of this peptide, we focused our studies on this protein using SkBr3 breast cancer cells, which are ideal for GPER evaluation. ERα17p inhibits cell growth by targeting membrane signaling. Identified as a GPER inverse agonist, it co-localizes with GPER and induces the proteasome-dependent downregulation of GPER. It also decreases the level of pEGFR (phosphorylation of epidermal growth factor receptor), pERK1/2 (phosphorylation of extracellular signal-regulated kinase), and c-fos. ERα17p is rapidly distributed in mice after intra-peritoneal injection and is found primarily in the mammary glands. The N-terminal PLMI motif, which presents analogies with the GPER antagonist PBX1, reproduces the effect of the whole ERα17p. Thus, this motif seems to direct the action of the entire peptide, as highlighted by docking and molecular dynamics studies. Consequently, the tetrapeptide PLMI, which can be claimed as the first peptidic GPER disruptor, could open new avenues for specific GPER modulators.