In this work, enhancement of daptomycin production by genome shuffling in Streptomyces roseosporus was conducted. Ultraviolet and NTG were used as mutagenizing agents to improve the volumetric productivity of the wild-type strain. Eight strains with enhanced daptomycin production were screened out as the starting population for genome shuffling. Daptomycin's production increased steadily with each round of genome shuffling. After the fourth round of fusion, a high-production strain (582 mg/L), named F4, was selected as a potential industrial production strain and its heredity stability was stable. Moreover, comparative analysis of the non-ribosomal peptide synthetase (NRPS) genes at the transcript level between the wild and the mutant was studied by RT-PCR in order to explore mechanism of genome shuffling. The transcript levels of NRPS genes dptA, dptBC, and dptD in the mutant were approximately 6.5 to 7 times higher than those in the wild. In summary, it is suggested that this strategy for increasing the daptomycin production in S. roseosporus by genome shuffling may provide an alternative approach to enhance the metabolite production in other Streptomyces.