A search for bacteriophage proteins with an EF-like calcium-binding loop, first discovered in the phage T5 endolysin (EndoT5), revealed a group of 71 peptidases of the M15_C subfamily. All the sequences found belonged to the phages infecting Gram-negative bacteria (Escherichia, Salmonella, Klebsiella, Shigella, Edwardsiella, Pectobacterium, Aeromonas, Shewanella etc.) In this work, we have characterized a hypothetical protein of Aeromonas bacteriophage 3, a representative of this group. It has been shown that the novel endolysin (EndoA3) is a functional peptidoglycan hydrolase, a Zn-containing Ca-dependent l-alanyl-d-glutamate peptidase (EC 3.4.24.B24). EndoA3 effectively lyses Gram-negative microorganisms, as well as Gram-positive bacteria of the genus Bacillus. It is a monomeric globular protein, and the formation of an active globule requires Zn in the catalytic and Ca in the regulatory centers. EndoA3 has a low affinity for Ca (K = 0.14 ± 0.01 mМ), insufficient for its activation in the cytoplasm of the host cell. Apparently, this mechanism of activation by periplasmic Ca emerged as a result of coevolution of bacteriophages and their Gram-negative.