The liver secretes hormones and growth factors, known as hepatokines, which influence metabolism in skeletal muscle and adipose tissue. Activin C and Activin E are two recently identified hepatokines that have been proposed to suppress lipolysis in adipose tissue via activation of the type I receptor, ALK7. Here, we set out to characterise the molecular mechanisms that govern Activin C synthesis and activity, and to generate more potent analogues for future in vivo experiments. First, we enhanced processing of the Activin C precursor by introducing a more efficient proprotein convertase cleavage site (RKKR). Importantly, enhanced processing corresponded with a dramatic increase in secreted Activin C activity. Next, we used site-directed mutagenesis to identify the residues in the pre-helix and alpha-helix of activin C involved in binding to ALK7. Subsequently, we modified these key receptor binding residues to generate a series of gain-of-function variants. The potency of these novel activin C analogues (EC50 0.3-0.6 ng/mL) was increased 10- or 20-fold, relative to wild-type Activin C (EC50 6 ng/mL). Treatment of ex vivo murine adipose tissue with highly potent Activin C analogues significantly reduced isoproterenol-induced lipolysis. Our study is the first to characterise Activin C residues involved in type I receptor binding and paves the way to characterise the role of the Activin C-ALK7 signalling axis in adipose tissue.