Rho is a hexameric helicase that induces bacterial transcription termination, but there has been a controversy about the mechanism of rho-dependent termination. In a conventional model called ‘RNA-dependent pathway’, rho first binds rut (rho-utilizing) site of RNA transcript, then chases RNA polymerase (RNAP) waiting on a pausing site, and finally disassembles the transcription complex. In a competing model called ‘RNAP-dependent pathway’, rho makes a stable complex with RNAP, and upon binding the rut site, induces termination through an allosteric mechanism. To clearly elucidate the mechanism of rho-dependent termination, we developed single-molecule fluorescence assays that can monitor the processes of rho-dependent termination of E. coli transcription. We found that whereas both RNA- and RNAP-dependent pathways operate for all tested rho-dependent termination sites with varying proportions, the RNAP-dependent pathway becomes more dominant as the pausing time at the termination site increases. This property of RNAP-dependent termination pathway makes it ideal for riboswitch-based regulation of rho-dependent termination as observed in the leader region of mgtA gene.