Coxiella burnetii is an intracellular pathogen that causes human Q fever, a disease that normally presents as a severe flu-like illness. Due to high infectivity and disease severity, the pathogen is considered a risk group 3 organism. Full-length lipopolysaccharide (LPS) is required for full virulence and disease by C. burnetii and is the only virulence factor currently defined by infection of an immunocompetent animal. Transition of virulent phase I bacteria with smooth LPS, to avirulent phase II bacteria with rough LPS, occurs during in vitro passage. Semi-rough intermediate forms are also observed. Here, the genetic basis of LPS phase conversion was investigated to obtain a more complete understanding of C. burnetii pathogenesis. Whole genome sequencing of strains producing intermediate and/or phase II LPS identified several common mutations in predicted LPS biosynthesis genes. After passage in broth culture for 30 weeks, phase I strains from different genomic groups exhibited similar phase transition kinetics and elevation of mutations in LPS biosynthesis genes. Targeted mutagenesis and genetic complementation using a new C. burnetii nutritional selection system based on lysine auxotrophy confirmed that six of the mutated genes were necessary for production of phase I LPS. Disruption of two of these genes in a C. burnetii phase I strain resulted in production of phase II LPS, suggesting inhibition of the encoded enzymes could represent a new therapeutic strategy for treatment of Q fever. Additionally, targeted mutagenesis of genes encoding LPS biosynthesis enzymes can now be used to construct new phase II strains from different genomic groups for use in pathogen-host studies at a risk group 2 level.