The Escherichia coli yqgF gene is highly conserved across a broad spectrum of bacterial genomes. The gene was first identified as being essential for cell growth during screening for targets for broad-spectrum antibiotics. YqgF is structurally similar to RuvC, a Holliday junction resolvase, but its function has not been established. This study describes the isolation of a temperature-sensitive yqgF mutant, the growth of which was inhibited by rho or nusA multicopy plasmids, indicating that YqgF is involved in transcription. Rho is a global transcription termination factor that acts at Rho-dependent terminator sites, which exist not only at the ends of genes but also within genes. The transcription of genes possessing intragenic, or upstream, Rho-dependent terminators was reduced in temperature-sensitive yqgF mutants. This transcription inhibition was sensitive to the Rho inhibitor, bicyclomycin. In addition, the transcription of mutant tnaA genes defective for upstream Rho-dependent termination was not significantly affected by the yqgF mutation. Taken together, these results suggest that YqgF is involved in anti-termination at Rho-dependent terminators in vivo.