Melanocortin-4 receptor (MC4R)-induced anorexigenic signaling in the hypothalamus controls body weight and energy homeostasis. So far, MC4R-induced signaling has been exclusively attributed to its coupling to G(s) proteins. In line with this monogamous G protein coupling profile, most MC4R mutants isolated from obese individuals showed a reduced ability to activate G(s). However, some mutants displayed enhanced G(s) coupling, suggesting that signaling pathways independent of G(s) may be involved in MC4R-mediated anorexigenic signaling. Here we report that the G(s) signaling-deficient MC4R-D90N mutant activates G proteins in a pertussis toxin-sensitive manner, indicating that this mutant is able to selectively interact with G(i/o) proteins. Analyzing a hypothalamic cell line (GT1-7 cells), we observed activation of pertussis toxin-sensitive G proteins by the wild-type MC4R as well, reflecting multiple coupling of the MC4R to G(s) and G(i/o) proteins in an endogenous cell system. Surprisingly, the agouti-related protein, which has been classified as a MC4R antagonist, selectively activates G(i/o) signaling in GT1-7 cells. Thus, the agouti-related protein antagonizes melanocortin-dependent G(s) activation not only by competitive antagonism but additionally by initiating G(i/o) protein-induced signaling as a biased agonist.