Coxiella burnetii is an intracellular bacterium responsible for the zoonotic disease Q fever, which causes both acute and chronic illness in humans. Until 2009 Coxiella was considered an obligate intracellular organism, hampering attempts to characterise its pathogenic mechanisms, but the publication of axenic culture methods has facilitated Coxiella research. In particular, the ability to culture Coxiella outside host cells has enabled us to optimise methodology for applying metabolomics to Coxiella, with the aim of understanding its central carbon metabolism (including essential carbon sources and pathways). Furthermore, metabolomics is a powerful tool for investigating the function of genes we have identified as required for intracellular replication. Here, we optimised techniques for bacterial cell quenching, metabolite extraction and analysis using both gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), and combined these techniques with stable isotope labelling, to elucidate functional pathways in Coxiella Nine Mile phase II strain RSA493 cultured under different conditions. We also used targeted metabolomics to investigate the function of genes, such as nadB, which we identified as dispensable for axenic growth but essential for growth inside HeLa cells. nadB encodes L- aspartate oxidase, which in other bacteria catalyses the conversion of L-aspartate into iminoaspartate, the first step in the prokaryotic nicotinamide adenine dinucleotide (NAD) synthetic pathway. Using both GC/MS and LC/MS we compared a nadB transposon mutant and wild type Coxiella, to compare metabolite levels both within the NAD synthetic pathway and more widely, to assess the broad effect of disrupting the synthesis of this important coenzyme and understand why this pathway is required for growth inside mammalian cells. This research is one of the first broad metabolomic studies of Coxiella, and demonstrates the usefulness of such studies in investigating both the overall metabolism of an organism, and the function of specific genes.