Methanogens are obligate anaerobes that form methane as a major product of their energy metabolism. They can elicit both specific and global regulatory responses to nutrient limitations. The hydrogenotrophic methanogen Methanococcus maripaludis possesses global responses to amino acids, H2, and nitrogen limitation but a specific response to phosphate limitation. The mechanisms underlying many of these regulatory responses are yet to be unraveled. A well-characterized phosphate-regulated promoter is a great candidate for developing a regulated gene expression system. This expression system will be crucial for implementing the expanding biotechnological potentials of Methanococcus maripaludis such as its capacity to produce methane and biomass from CO2 or provide vital biocatalysts on an industrial scale.A library of differentially regulated promoters, which responds to inorganic phosphate concentration in the growth medium was designed and characterized based on the promoter of a phosphate transporter in M. maripaludis. This regulated system increases heterologous gene expression by 4 to 6-fold when the medium phosphate drops to growth-limiting concentrations. Hence, growth is decoupled from heterologous gene expression without the need of adding an inducer. The minimal pst promoter was identified and contains a conserved AT-rich region for phosphate-dependent regulation. Rational changes to the transcription start site and 5' untranslated region resulted in differential protein production with regulation remaining intact. Compared to a previous expression system based upon the histone promoter, this regulated expression system resulted in significant improvements in expression. The phosphate regulatory protein (PrpX) is a PhoU homolog with a winged helix-turn-helix DNA binding domain and a PhoU domain that potentially serves as a sensory or phosphate-binding domain. Deletion of prpX results in loss of phosphate-dependent regulation from the pst promoter and an intermediate expression level irrespective of the phosphate concentration. Binding experiments showed that PrpX can specifically bind pst promoter at the conserved AT-rich region and the presence of phosphate up to 200-fold excess of the protein resulted in increased DNA binding. Our proposed model of regulation is that PrpX acts as a transcriptional regulator, capable of repressing or inducing expression from the pst promoter based on conformational changes resulting from binding phosphate.