A cytoplasmic protein FROUNT serves as a common regulator of chemokine receptors CCR2 and CCR5 (Nat Immunol. 2005, 6:827, J Immunol. 2009, 183:6387), both of which are involved in macrophage accumulation in inflammation and cancer. We have identified a binding site for FROUNT conserved among these receptors and the interaction mechanism between FROUNT and the receptors (Biochem J. 2014, 457:313). We narrowed down the region of CCR2, a family of seven-transmembrane G protein-coupled receptors, responsible for FROUNT binding by yeast two-hybrid analysis. The identified region of CCR2 was located in the putative helix 8, or membrane-proximal carboxy-terminal intracellular region. We call this region ‘CCR2 Pro-C’. CCR2 Pro-C was predicted to form an amphipathic helix structure, which was confirmed by NMR analysis. Synthesized peptides corresponding to the CCR2 Pro-C sequence directly interacted with FROUNT in vitro. Leu316Thr mutation in the hydrophobic side of the predicted helix decreased the affinity for FROUNT. The hydrophobic side, but not the hydrophilic side, of the Pro-C region is conserved among FROUNT-binding receptors. These results suggest that the hydrophobic side of the Pro-C is a responsible element for FROUNT binding. We examined this in the context of full-length CCR2 in cells. Co-immunoprecipitation assays revealed that CCR2-Leu316Thr mutation diminished the interaction between FROUNT and CCR2 in cells. Furthermore, this mutation into the FROUNT-binding element in receptor impaired cellular chemotaxis. These findings provide a novel approach for regulating the chemokine receptor signaling mediated by the cytosolic regulator FROUNT.