FK506 is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces tsukubaensis. However, the production capacity of the strain is very low. To improve production, atmospheric and room temperature plasma (ARTP) mutagenesis was adopted to get the initial strains used in genome shuffling (GS). After three rounds of GS, S. tsukubaensis R3-C4 was the most productive strain, resulting in a FK506 concentration of 335 mu g/mL, 2.6 times than that of the original wild-type strain. Moreover, exogenous DMSO 4% (v/v) addition could induce efflux of FK506 and increased FK506 production by 27.9% to 429 mu g/mL. Finally, analyses of the differences in morphology, fermentation characteristics and specific gene expression levels between S. tsukubaensis R3-C4 and the wild-type strain revealed that R3-C4 strain: has hampered spore differentiation, thicker mycelia and more red pigment, which are likely related to the downregulation of bldD and cdgB expression. In addition, the expression levels of fkbO, fkbP, dahp, pccB and prpE all showed up-regulation at diverse degrees compared to the wild-type S. tsukubaensis. Overall, these results show that a combined approach involving classical random mutation and exogenous feeding can be applied to increase FK506 biosynthesis and may be applied also to the improvement of other important secondary metabolites.