Four kinds of 2',3'-cNMPs have been identified in animals and plants, and their physiological roles are also suggested. However, in prokaryotes, while 2',3'-cCMP and 2',3'-cUMP are reported, 2',3'-cGMP or 2',3'-cAMP have never been identified from bacteria or archaea. In addition, there has been no biological method to prepare these cyclic nucleotides. During the study of a novel gene LfliZ from deep-sea bacterium Pseudoalteromonas sp. SM9913, we found that the recombinant LfliZ oligomers contained endogenous substrates, which were then identified to be 2',3'-cCMP, 2',3'-cUMP, 2',3'-cGMP, and 2',3'-cAMP, showing the first evidence for the biological existence of four kinds of 2',3'-cNMPs in prokaryotes. In addition, we further developed a biological method to simultaneously prepare four kinds of 2',3'-cNMPs from Escherichia coli through expressing LfliZ.