Vibrio choleraecauses the human disease cholera by producing a potent toxin. TheV. choleraevirulence pathway involves an unusual transcription step: the bitopic inner-membrane proteins TcpP and ToxR activatetoxTtranscription. As ToxT is the primary direct transcription activator inV. choleraepathogenicity, its regulation by membrane-localized activators is key in the disease process. However, the molecular mechanisms by which membrane-localized activators engage the transcription process have yet to be uncovered in live cells. Here we report the use of super-resolution microscopy, single-molecule tracking, and gene knockouts to examine the dynamics of individual TcpP proteins in liveV. choleraecells with < 40 nm spatial resolution on a 50 ms timescale. Single-molecule trajectory analysis reveals that TcpP diffusion is heterogeneous and can be described by three populations of TcpP motion: one fast, one slow, and one immobile. By comparing TcpP diffusion in wild-typeV. choleraeto that in mutant strains lacking eithertoxRor thetoxTpromoter, we determine that TcpP mobility is greater in the presence of its interaction partners than in their absence. Our findings support a mechanism in which ToxR recruits TcpP to thetoxTpromoter for transcription activation.