Aims: To improve lincomycin A production and decrease the content of byproduct lincomycin B in an industrial lincomycin-producing strain. Methods and Results: The in silico analysis indicated that LmbW could be involved in propylproline biosynthesis of lincomyin A. In this study, we constructed an lmbW deletion mutant and found that the mutant lost the ability to produce lincomycin A, but increased the accumulation of lincomycin B. The loss of lincomycin A production can be restored by complementing the mutant with the expression of lmbW gene. When lmbW and metK (encoding S-adenosylmethionine synthetase) was co-overexpressed, lincomycin A titre was 1744.6 mg l(-1), a 35.83% improvement over the original strain. Meanwhile, the content of lincomycin B was reduced to 4.41%, a remarkable decrease of 34.76%, compared to that of the original strain. Conclusions: lmbW encodes a C-methyltransferase involved in the biosynthesis of lincomycin A but not lincomycin B. Co-overexpression of lmbW and metK improved lincomycin A production and decreased the content of lincomycin B. Significance and Impact of the Study: The engineered Streptomyces lincolnensis strain shows promising application in the fermentation production of lincomycin A, which may help cut production costs and simplify downstream separation processes.