Objective To identify whether the BLJ_864 and BLJ_865 in the chromosome of Bifidobacterium longum JDM301 (B.longum JDM301) constitute a type II toxinantitoxin system (TA system) of mazEF family. Methods The co-transcription of BLJ_864 and BLJ_865 in B.longum JDM301 and E.coli was analyzed by the RT-PCR. The co-expression of BLJ_864 and BLJ_865 encoded antitoxin protein and toxin protein in the intact operon that carried BLJ_864 and BLJ_865 was proved by transforming the plasmid, which carried the operon, into the E.coli strain. The inhibition effect of BLJ_864 encoded toxin protein on the growth of E.coli cells and the corresponding effect of BLJ_865 encoded antitoxin protein on removing the inhibition effect were identified by the expression of BLJ_864 encoded toxin protein or co-expression of the toxin protein and BLJ_865 encoded antitoxin protein in E.coli. Results BLJ_864 and BLJ_865 constituted a dual operon and could co-transcript in the natural strain of B.longum JDM301. BLJ_864 encoded toxin protein and BLJ_865 encoded antitoxin protein could co-express in the heterologous host of E.coli when induced by isopropyl-β-D-thiogalactopyranoside (IPTG). The expression of BLJ_864 encoded toxin protein inhibited the growth of E.coli, while the co-expression of BLJ_864 encoded toxin protein and BLJ_865 encoded antitoxin protein could antagonize the inhibition effect of toxin protein. Conclusion BLJ_864 and BLJ_865 in the chromosome of B.longum JDM301 encode a functional type II TA system of mazEF family.