Because the aquatic environments contain countless kinds of bacteria and virus and fishes are armed with less developed adaptive immune system comparing with mammals, one may expect the innate immunity including the complement system of fish plays much more important roles in defense against pathogen invading. The site-models tested on fish and mammalian C3 genes revealed that these two groups of vertebrates which are flourishing in the aquatic and terrestrial environments, respectively, experienced different evolutionary patterns. No evidence of positive selection was detected in mammalian C3 while seven sites were found to be under positive selection in fish C3, indicating the different evolutionary pressure on these two groups whose living environments differed hugely. Molecular evolution analyses were also conduced to explore the possible evolutionary process of C3. Many positively selected sites were detected among the common ancestral lineages to the vertebrates, mammals and protacanthopterygian and ostariophysian fishes, indicating that episodic positive selection events had happened during the C3 evolution along these lineages. The first period of positive selection happened with the emergence of vertebrates. From the evolutionary standpoint, the complement system is present in both of vertebrates and a wide range of invertebrates. Unlike the vertebrates, the complement system of invertebrate was more primitive although they showed some complexity and diversity. Those invertebrate complement systems lack the antibody and thus the classical pathway, which is based on the antibody-recognizing activation cascade, and seem to represent a prototypic opsonin system composed of C3 and its activation cascades that seem to correspond to mammalian lectin and/or alternative pathways. The ancient origin of C3 gene can be traced back to cnidarians, one of the most primitive metazoan members and it has been evolutionarily retained in both deuterostomes and some lineages of protostome, such as arthropods and mollusks. The antimicrobial activities of the invertebrate C3, through a complement-mediated phagocytosis, have been proven only in the sea urchin and ascidians. And no evidence of MK-1775 direct cytolytic activity has been proven in invertebrate primitive complement system. Thus, we speculate that with the evolvement of antibody in the vertebrate, the complement system had experienced the first period of positive selection on the ancestral vertebrates to evolve the classical pathway of C3-activation and the cytolytic pathway. These huge advances of immunity, emergence of antibody and the classical pathway of activating complement system, promoted the flourish of the ancestral vertebrates. The second period of positive selection happened on the early period of fish evolutionary history. The ancestral lineage leading to ostariophysian and protacanthopterygian fish also showed positively selected sites, indicating one more period of positive selection event on C3. Besides that, six positive selection sites were also detected among the ancestral lineage leading to all actinopterygian fish.