ABSTRACT A fundamental aspect of most infectious diseases is the need for the invading microbe to proliferate in the host. However, little is known about the metabolic pathways required for pathogenic microbes to colonize and persist in their hosts. In this study, we used RNA sequencing (RNA-seq) to generate a high-resolution transcriptome of the opportunistic pathogen Aggregatibacter actinomycetemcomitans in vivo . We identified 691 A. actinomycetemcomitans transcriptional start sites and 210 noncoding RNAs during growth in vivo and as a biofilm in vitro . Compared to in vitro biofilm growth on a defined medium, ∼14% of the A. actinomycetemcomitans genes were differentially regulated in vivo . A disproportionate number of genes coding for proteins involved in metabolic pathways were differentially regulated in vivo , suggesting that A. actinomycetemcomitans in vivo metabolism is distinct from in vitro growth. Mutational analyses of differentially regulated genes revealed that formate dehydrogenase H and fumarate reductase are important A. actinomycetemcomitans fitness determinants in vivo . These results not only provide a high-resolution genomic analysis of a bacterial pathogen during in vivo growth but also provide new insight into metabolic pathways required for A. actinomycetemcomitans in vivo fitness.