Abstract Nisin is a natural peptide used as a preservative in a variety of food products, in which it inhibits mainly Gram-positive bacterial growth, including multidrug-resistant pathogens. However, its application range depends on the cost-effective production and purification of this molecule. Our group has previously produced nisin by Lactococcus lactis cultivation in milk whey, which is an industrial residue from dairy production. To our knowledge, no report used milk whey as a culture medium, although several investigators have purified nisin using different techniques. We thus aimed to establish a low-cost purification of nisin obtained by this process. Samples were diluted in ammonium sulphate, applied onto \HIC\ columns (butyl sepharose \CL\ 4B matrix), and eluted with Milli-Q water or PBS. Elution fractions were monitored for protein content and nisin antibacterial activity. Water elution resulted in purification factor values (270, commercial nisin; 775, nisin produced in-house) higher than those obtained with \PBS\ elution. We concluded that purification of nisin does not require precipitation with ammonium sulphate, therefore allowing step/cost reduction. Moreover, purification from milk whey using \HIC\ provides nisin with high activity and low salt content, which can further be applied to a variety of areas.