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Prodigiosin is a secondary metabolite produced in several species of bacteria. It exhibits antimicrobial and anticancer properties. Methods for the extraction and identification of prodigiosin and their related derivatives from bacterial cultures typically depend on solvent-based extractions followed by NMR spectroscopy. The estuarine bacterium, V. gazogenes PB1, was previously shown to produce prodigiosin. This conclusion, however, was based on analytical data obtained from ultraviolet-visible absorption spectrophotometry and infrared spectroscopy. Complete dependence on these techniques would be considered inadequate for the accurate identification of the various members of the prodiginine family of compounds, which possess very similar chemical structures and near-identical optical properties. In this study, we extracted prodigiosin from a culture of Vibrio gazogenes PB1 cultivated in minimal media, and for the first time, confirmed the synthesis of prodigiosin Vibrio gazogenes PB1 using NMR techniques. The chemical structure was validated by [sup.1]H and [sup.13]C NMR spectroscopy, and further corroborated by 2D NMR, which included [sup.1]H-[sup.1]H-gDQFCOSY, [sup.1]H-[sup.13]C-gHSQC, and [sup.1]H-[sup.13]C-gHMBC, as well as [sup.1]H-[sup.1]H-homonuclear decoupling experiments. Based on this data, previous NMR spectral assignments of prodigiosin are reaffirmed and in some cases, corrected. The findings will be particularly relevant for experimental work relating to the use of V. gazogenes PB1 as a host for the synthesis of prodigiosin.