Corynebacterium glutamicum ATCC13032 and Brevibacterium flavum JV16 were engineered for L-valine production by over-expressing ilvEBN ( r ) C genes at 31 °C in 72 h fermentation. Different strategies were carried out to reduce the by-products' accumulation in L-valine fermentation and also to increase the availability of precursor for L-valine biosynthesis. The native promoter of ilvA of C. glutamicum was replaced with a weak promoter MPilvA (P-ilvAM1CG) to reduce the biosynthetic rate of L-isoleucine. Effect of different relative dissolved oxygen on L-valine production and by-products' formation was recorded, indicating that 15 % saturation may be the most appropriate relative dissolved oxygen for L-valine fermentation with almost no L-lactic acid and L-glutamate formed. To minimize L-alanine accumulation, alaT and/or avtA was inactivated in C. glutamicum and B. flavum, respectively. Compared to high concentration of L-alanine accumulated by alaT inactivated strains harboring ilvEBN ( r ) C genes, L-alanine concentration was reduced to 0.18 g/L by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ( r ) C, and 0.22 g/L by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ( r ) C. Meanwhile, L-valine production and conversion efficiency were enhanced to 31.15 g/L and 0.173 g/g by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ( r ) C, 38.82 g/L and 0.252 g/g by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ( r ) C. This study provides combined strategies to improve L-valine yield by minimization of by-products' production.