The E. coli 6S RNA was discovered more than three decades ago, yet its function has remained elusive. Here, we demonstrate that 6S RNA associates with RNA polymerase in a highly specific and efficient manner. UV crosslinking experiments revealed that 6S RNA directly contacts the σ⁷⁰ and β/β′ subunits of RNA polymerase. 6S RNA accumulates as cells reach the stationary phase of growth and mediates growth phase–specific changes in RNA polymerase. Stable association between σ⁷⁰ and core RNA polymerase in extracts is only observed in the presence of 6S RNA. We show 6S RNA represses expression from a σ⁷⁰-dependent promoter during stationary phase. Our results suggest that the interaction of 6S RNA with RNA polymerase modulates σ⁷⁰-holoenzyme activity.