Interactions between biomolecules are difficult to observe directly due to the small size scales involved. Super-resolution microscopy offers a glimpse of such dynamics beyond the diffraction limit of light, even inside living cells. We use single-molecule fluorescence microscopy to examine the nanometer-scale movements of TcpP, a membrane-bound transcription activator, in the human pathogen Vibrio cholerae. TcpP, along with a second bitopic inner-membrane protein, ToxR, activates transcription of ToxT, the primary transcription activator for cholera toxin production.Here we track TcpP molecules tagged with the red photoactivatable fluorescent protein PA-mCherry with a spatial resolution of 30 nm and a temporal resolution of 50 ms, revealing interactions among TcpP, ToxR and the toxT promoter. Through analysis of single-molecule trajectories based on mean square displacements and cumulative probability distributions of step sizes, we have revealed three modes of TcpP motion: a “fast” population, a “slow” population, and an immobile population. Furthermore, by comparing TcpP dynamics in TcpP-PA-mCherry fusion cells with the motion of TcpP in ToxR knockout cells, we demonstrate that TcpP moves faster in the presence of ToxR. Surprisingly, we discover that the effect on TcpP dynamics of knocking out the toxT promoter is very similar to the effect of knocking out ToxR, implying that the promoter plays a role in the interaction between ToxR and TcpP. These results support a mechanism in which ToxR binds DNA and recruits TcpP to the toxT promoter site.