<p>Caspases are cysteine-dependent aspartate-directed proteases which have critical functions in programmed cell death and inflammation. Their catalytic activity depends on a catalytic dyad of cysteine and histidine within a characteristic protein fold, the so-called caspase domain. Here, we investigated the evolution of caspase-16 (CASP16), an enigmatic member of the caspase family, for which only a partial human gene had been reported previously. The presence of <i>CASP16</i> orthologs in placental mammals, marsupials and monotremes suggests that caspase-16 originated prior to the divergence of the main phylogenetic clades of mammals. Caspase-16 proteins of various species contain a carboxy-terminal caspase domain and an amino-terminal prodomain predicted to fold into a caspase domain-like structure, which is a unique feature among caspases known so far. Comparative sequence analysis indicates that the prodomain of caspase-16 has evolved by the duplication of exons encoding the caspase domain, whereby the catalytic site was lost in the amino-terminal domain and conserved in the carboxy-terminal domain of caspase-16. The murine and human orthologs of <i>CASP16</i> contain frameshift mutations and therefore represent pseudogenes (<i>CASP16P</i>). <i>CASP16</i> of the chimpanzee displays more than 98% nucleotide sequence identity with the human <i>CASP16P</i> gene but, like <i>CASP16</i> genes of other primates, has an intact protein coding sequence. We conclude that caspase-16 structurally differs from other mammalian caspases, and the pseudogenization of <i>CASP16</i> distinguishes humans from their phylogenetically closest relatives.</p>