Melanotan-II primarily exerts its effects by binding to the melanocortin-1 receptor (MC1R), a G protein-coupled receptor widely distributed on the surface of cutaneous melanocytes.

Upon binding to MC1R, Melanotan-II activates G proteins, which in turn stimulate adenylyl cyclase, leading to elevated intracellular levels of cyclic adenosine monophosphate (cAMP). Acting as a second messenger, cAMP activates protein kinase A (PKA); PKA then promotes tyrosinase gene expression and tyrosinase activity by phosphorylating a series of downstream target proteins. Tyrosinase is a key enzyme in melanogenesis, catalyzing the stepwise oxidation of tyrosine to DOPA and subsequently to melanin. Additionally, Melanotan-II may influence melanocyte proliferation, differentiation, and melanin synthesis and secretion by modulating other signaling pathways, such as the phospholipase C (PLC)–protein kinase C (PKC) pathway.

Beyond its effects on melanocytes, Melanotan-II has been found to modulate sexual behavior. The underlying mechanisms likely involve the regulation of neurotransmitters (such as dopamine and nitric oxide) within the central nervous system, though further research is required to fully elucidate these processes.