Significance GDF8 is a signaling protein that inhibits muscle mass. Inhibitors of GDF8 are highly sought as therapeutics for the treatment of muscle-wasting diseases. During synthesis, GDF8 is made as a precursor where the signaling segment is cleaved from the N-terminal prodomain, which remains associated and inhibits signaling. Activation involves an additional cleavage of the prodomain. We demonstrate GDF8 signaling could be gained through a conformational change where the prodomain remains associated with the signaling segment. Alteration of the prodomain can weaken the interactions causing GDF8 to signal, thus alleviating inhibition by the prodomain. This study illuminates how GDF8 transitions from an inhibited state to an active state—information that will help to understand the mechanism of GDF8 signaling.