GPR15 is a G protein-coupled receptor (GPCR) that directs lymphocyte homing to the colon and skin. Recent studies have identified a chemokine-like protein C10orf99/GPR15L as a functional ligand of GPR15. In this study we examined the structural elements that regulate the GPR15-GPR15L interaction with primary focus on post-translational modifications (PTMs) of receptor N-terminus and on the C-terminus of the ligand. Our findings reveal that the GPR15 receptor is sulfated on the N-terminal Tyr residue(s) and disruption of Tyr sulfation inhibited binding of GPR15L. In contrast, the disruption of O-glycosylation on the N-terminal Thr/Ser residues or the removal of α2,3-linked sialic acids from O-glycans enhanced the GPR15L binding. Thus, GPR15 represents a unique chemoattractant receptor in which different N-terminal PTMs regulate its ligand binding in a contrasting manner. We further demonstrate that unlike canonical chemokines, GPR15L activity critically requires its extreme C-terminal residue and its hydrophobicity may be a key attribute that facilitates an optimal interaction with the receptor. Our results reveal novel insights into chemoattractant receptor-ligand interaction and provide a valid footing for potential intervention targeting GPR15-GPR15L axis.