Preparative high-speed counter-current chromatography (HSCCC) was successfully applied to the isolation and purification of two macrolactin antibiotics from marine bacterium Bacillus amyloliquefaciens for the first time using stepwise elution with a pair of two-phase solvent systems composed of n-hexane–ethyl acetate–methanol–water at (1:4:1:4, v/v) and (3:4:3:4, v/v). The preparative HSCCC separation was performed on 300mg of crude sample yielding macrolactin B (22.7mg) and macrolactin A (40.4mg) in a one-step separation, with purities over 95% as determined by HPLC. The structures of these compounds were identified by MS, 1 H NMR and 13 C NMR. Our results demonstrated that HSCCC was an efficient technique to separate marine antibiotics, which provide an approach to solve the problem of their sample availability for drug development.