Ribosome elongation factor G encoded by fusA promotes the translocation step of protein synthesis in bacteria; ribosome recycling factor encoded by frr, together with the elongation factor G, dissociates ribosomes from messenger RNA after the termination of translation. Both factors play important roles during protein synthesis in bacteria. In this study, we found that overexpression of fusA and/or frr led to the increase of l-isoleucine production in Corynebacterium glutamicum IWJ001, an l-isoleucine production strain generated by random mutagenesis. Reverse transcription polymerase chain reaction analysis showed that transcriptional levels of genes lysC, hom, thrB, ilvA, ilvBN, and ilvE encoding the key enzymes in the biosynthetic pathway of l-isoleucine increased in C. glutamicum IWJ001 when fusA and/or frr were overexpressed. Co-overexpression of fusA and frr, together with genes ilvA, ilvB, ilvN, and ppnk in C. glutamicum IWJ001, led to 76.5 % increase of l-isoleucine production in flask cultivation and produced 28.5 g/L l-isoleucine in 72-h fed-batch fermentation. The results demonstrate that overexpressing ribosome elongation factor G and ribosome recycling factor is an efficient approach to enhance l-isoleucine production in C. glutamicum.