The melanocortin-3 receptor (MC3R) is a G protein-coupled receptor involved in regulating energy metabolism, cardiovascular function, and inflammation. To gain a better understanding of the structure-function relationship of the MC3R, we used alaninescanning mutagenesis to investigate the functions of residues 247–273 in the third intracellular loop (ICL3) of the human MC3R (hMC3R). Ligand binding and signaling parameters of the mutants were measured. The results showed that six mutants at the N terminus of ICL3 had decreased receptor occupancy (an estimate of relative binding capacity) whereas six mutants at the C terminus of ICL3 had increased receptor occupancy. M247A, R252A, H254A, and K256A had decreased maximal responses (M247A also had increased EC50) whereas F250A, P262A, and H272A had increased maximal responses. None of the mutants was constitutively active. The binding and signaling properties of the other mutants were not significantly different from that of the wild type hMC3R. In summary, we presented detailed functional data on the functions of the residues in ICL3 of hMC3R, providing important constraints for modeling ligand binding and G protein coupling/activation in the hMC3R.