Gene transcription occurs in short bursts interspersed with silent periods, and these kinetics can be altered by promoter structure. The effect of alternate promoter architecture on transcription bursting is not known. We studied the human prolactin (hPRL) gene that contains two promoters, a pituitary-specific promoter that requires the transcription factor Pit-1, and displays dramatic transcriptional bursting activity, and an alternate upstream promoter that is active in non-pituitary tissues. We studied large hPRL genomic fragments with luciferase reporters, and used bacterial artificial chromosome (BAC) recombineering to manipulate critical promoter regions. Stochastic switch mathematical modelling of single-cell time-lapse luminescence image data revealed that the Pit-1-dependent promoter showed longer, higher-amplitude transcriptional bursts. Knockdown studies confirmed that the presence of Pit-1 stabilised and prolonged periods of active transcription. Pit-1 therefore plays an active role in establishing the timing of transcription cycles, in addition to its cell-specific functions.