Circular bacteriocins are potent antimicrobials against pathogenic Gram-positives. In searching for marine bacteriocins, an antibacterial peptide (flexusin A) was purified from the fermentation broth of marine bacterium R29-2. Genome sequencing and gene annotation revealed the chromosome contained an unknown circular bacteriocin gene cluster. Approaches including shot-gun proteomics analysis, AntiSMASH and BAGEL4 predication as well as the comprehensive sequence alignment, were then conducted, respectively, to verify the correlation of flexusin A with the gene-encoded precursor peptide. The results confirmed that flexusin A was the mature circular bacteriocin of the predicated precursor peptide with six amino acids as leader peptide. Flexusin A was 6098.4 Da in size, with a net charge of +3 and PI of 9.60. It shared the typical saposin-like fold spatial conformation features as commonly found in other circular bacteriocins. Flexusin A was pH, thermal, and protease tolerant. It exhibited a narrow antimicrobial spectrum against Gram-positives, and it can strongly inhibit by causing cell destruction via membrane destabilization. Taken together, a novel circular bacteriocin flexusin A was identified in this work. The characterization of flexusin A has extended circular bacteriocins family to 26 members. This is also the first report on bacteriocin production by .