As a housekeeping gene, glyceraldehyde 3phosphate dehydrogenase (GAP) is ubiquitously expressed in various microorganisms. Therefore, its promoter is believed a strong constitutive promoter. In this study, a novel PGAP was predicted and cloned from Bifidobacterium bifidum. The strength of this promoter in both Escherichia coli and bifidobactteria was probed by enzyme activity assay using βglucuronidase (gusA) as reporter. Furthermore, impacts of different carbon sources and derivatives of GAP substrate on the strength of promoter in bifidobacteria were tested. Sugar fermentation experiments show the promoter is strongest when glucose was used as carbon source, and supplementation of three kinds of substrates influences the strength as well. Site directed mutagenesis (SDM) of predicted 10 region yielded three derivatives with different strengths. The mutant 3, namely pMGAP3 shows the strongest activity, which has more close structure to the consensus sequence of sigma 70 like promoters. In the end, alignment of all representative PGAP sequences in bifidobacteria demonstrates they are very conservative in some core regions, like TATA box, transcription start site (TSS), and ribosome binding site (RBS).