Cystatins, the cysteine protease inhibitors, and the cathelin-like domain (CLD) of the antimicrobial cathelicidins are classified into the same superfamily because of their overall structural similarity. However, their evolutionary relationship has remained obscure owing to low sequence similarity. Structural similarity of two proteins often provides evidence for divergent evolution; however, structural convergence can not be completely ruled out in this case. Conserved gene structure and related function provide new evidence in favor of a common ancestral origin for cystatins and CLDs. On the basis of two observations, the C-terminal location of the cathelicidin antimicrobial domain and evolutionary gain of one 3' intron, I propose a gradual evolution model to explain how the AMD evolved from the ancestral cystatin scaffold.