Transcription termination factor rho is a hexameric, RNA-dependent NTPase that can adopt active closed-ring and inactive open-ring conformations. The Sm-like protein Rof, a homolog of the RNA chaperone Hfq, inhibits rho-dependent termination in vivo but recapitulation of this activity in vitro has proven difficult and the precise mode of Rof action is presently unknown. Here, our cryo-EM structures of rho-Rof and rho-RNA complexes show that Rof undergoes pronounced conformational changes to bind rho at the protomer interfaces, undercutting rho conformational dynamics associated with ring closure and occluding extended primary RNA-binding sites that are also part of interfaces between rho and RNA polymerase. Consistently, Rof impedes rho ring closure, rho-RNA interactions and rho association with transcription elongation complexes. Structure-guided mutagenesis coupled with functional assays confirms that the observed rho-Rof interface is required for Rof-mediated inhibition of cell growth and rho-termination in vitro. Bioinformatic analyses reveal that Rof is restricted to Pseudomonadota and that the rho-Rof interface is conserved. Genomic contexts of rof differ between Enterobacteriaceae and Vibrionaceae, suggesting distinct modes of Rof regulation. We hypothesize that Rof and other cellular anti-terminators silence rho under diverse, but yet to be identified, stress conditions when unrestrained transcription termination by rho may be detrimental.