An RpoS factor is a transcriptional regulator which participates in numerous biological processes. In this work, we investigated the transcriptional regulation of proBA and proC composing proline biosynthetic pathway in Escherichia coli. While the proBA and proC genes were greatly induced in an exponential growth phase, they were dramatically repressed in a stationary growth phase in the wild type E. coli. Unlike the wild type E. coli, the proBA and proC genes were not repressed even in the stationary growth phase in its isogenic rpoS mutant. These results suggest that the RpoS factor acts as a transcriptional repressor of proBA and proC genes. The production of threonine, methionine, lysine, and arginine in the rpoS mutant were also increased by more than two times compared to its parental wild type, suggesting that the mutant is able to be used as an useful host strain for the amino acid overproduction.