The butyrate production by engineered Escherichia coli is afflicted by both low titer and low selectivity (defined as the butyrate/acetate (B/A) ratio). To address this issue, a strategy for metabolic engineering of E. coli was implemented including (1) elimination of all major NADH-dependent reactions in the fermentation metabolism, (2) reconstruction of a heterologous pathway leading to butyryl-CoA, (3) recruitment of endogenous atoDA for conversion of butyryl-CoA to butyrate with acetate as a CoA acceptor, and (4) removal of the acetate-synthesis pathway. Grown on glucose (20 g/L) plus acetate (8 g/L), the engineered strain consumed almost all glucose and acetate and produced 10 g/L butyrate as a predominant product within 48 h. It leads to high butyrate selectivity with the B/A ratio reaching 143. The result shows that our proposed approach may open a new avenue in biotechnology for production of butyrate in E. coli.