Factor Rho is key factor in factor-dependent termination in prokaryote. Rho-dependent termination happens in the regions identified as Rho-terminator, many of which position in downstream of open reading frame (ORF). In recent, it was reported that Rho-terminator locates the leader region of ORF and that some of them shows varying termination efficiency depending on the change of external environment. The representative case is mgtA terminator, combined with magnesium-sensing riboswitch only to regulates termination efficiency. To study the regulation mechanism of Rho-dependent premature transcription termination, we developed single-molecule fluorescence assay for observing transcription termination. We found that there are two distinct termination pathways, RNAP-dependent pathway and RNA-dependent pathway in mgtA terminator. RNAP-dependent pathway solely includes termination caused by RNAP-bound Rho, but RNA-dependent pathway means termination caused by factor Rho directly binding to the RNA. RNAP-dependent pathway showed more dependence on magnesium condition than RNA-dependent pathway, which means RNAP-dependent pathway mainly bring about magnesium-sensing property of mgtA. To understand the difference in sensitivity to ion of two pathways, we examined several different DNA substrates with mutants in pausing site. These examinations showed that RNAP-dependent pathway was slower than RNA-dependent pathway, so this pathway more sensitively influenced by b narrower time window in disruption of pausing site. These results suggested that slow termination pathway caused by RNAP-bound Rho finely tune premature transcription termination in response to external change.