We have performed a transcriptomic in vivo study with Enterococcus faecalis OG1RF in the intestine of living mice to identify novel latent and adaptive fitness determinants within E. faecalis. From 2,658 genes that are present in E. faecalis strain OG1RF, 124 genes were identified as significantly differentially expressed within the intestinal tract of living mice as compared to exponential growth in BHI broth. The groups of significantly up- or down-regulated genes consisted of 94 and 30 genes, respectively, for which 46 and 18 a clear annotation to a functionally described protein was found. These included genes involved in energy metabolism (e.g., dhaK and glpK pathway), transport and binding mechanisms (e.g., phosphoenolpyruvate carbohydrate PTS) as well as fatty acid metabolism (fab genes). The novel putative fitness determinants found in this work may be helpful for future studies of E. faecalis adaptation to the intestinal tract, which is also a prerequisite for infection in a compromised or inflamed host.