The melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis and has emerged as a premier target for obesity treatment. Numerous mutations in transmembrane domain 6 (TM6) of MC4R resulting in functional alterations have been identified in obese patients. Several mutagenesis studies also provided some data suggesting the importance of this domain in receptor function. To gain a better understanding of the structure-function relationship of the receptor, we performed alanine-scanning mutagenesis in TM6 to determine the functions of side chains. Of the 31 residues, two were important for cell surface expression, five were indispensable for α-melanocyte-stimulating hormone (α-MSH) and β-MSH binding, and six were important for signaling in the Gs-cAMP-PKA pathway. H264A, targeted normally to the plasma membrane, was undetectable by competitive binding assay and severely defective in basal and stimulated cAMP production and ERK1/2 phosphorylation. Nine mutants had decreased basal cAMP signaling. Seven mutants were constitutively active in cAMP signaling and their basal activities could be inhibited by two MC4R inverse agonists, Ipsen 5i and ML00253764. Five mutants were also constitutively active in the MAPK pathway with enhanced basal ERK1/2 phosphorylation. In summary, our study provided comprehensive data on the structure-function relationship of the TM6 of MC4R. We identified residues that are important for cell surface expression, ligand binding, cAMP generation, and residues for maintaining the WT receptor in active conformation. We also reported constitutive activation of the MAPK pathway and biased signaling. These data will be useful for rationally designing MC4R agonists and antagonists for treatment of eating disorders.