The Escherichia coli RNase G is known as an endoribonuclease responsible for the 5'-end maturation of 16S rRNA and degradation of several specific mRNAs such as adhE and eno mRNAs. In this study, we found that an RNase G mutant derived from the MC1061 strain did not grow on a glucose minimal medium. Genetic analysis revealed that simultaneous defects of cra and ilvIH, encoding a transcriptional regulator of glycolysis/gluconeogenesis and one of isozymes of acetohydroxy acid synthase, respectively, were required for this phenomenon to occur. The results of additional experiments presented here indicate that the RNase G mutation, in combination with cra mutation, caused the increased production of pyruvic acid from glucose, which was then preferentially converted to valine due to the ilvIH mutation, resulting in depletion of isoleucine. In fact, the rng cra double mutant produced increased amount of pyruvate in the medium. These results suggest that the RNase G mutation could be applied in the breeding of producer strains of pyruvate and its derivatives such as valine.