Enterocin NKR-5-3B, one of the multiple bacteriocins produced by Enterococcus faecium NKR-5-3, is a 64 aa novel circular bacteriocin that displays broad-spectrum antimicrobial activity. Here we report the identification, characterization, and three-dimensional NMR solution structure determination of enterocin NKR-5-3B. Enterocin NKR-5-3B is characterized by four helical segments that enclose a compact hydrophobic core, which together with its circular backbone, impart high stability and structural integrity. We also report the corresponding structural gene, enkB, which encodes an 87 aa precursor peptide that undergoes a yet to be described enzymatic processing that involves adjacent cleavage and ligation of Leu24 and Trp87 to yield the mature (circular) enterocin NKR-5-3B.