Enterococcus faecium is a commensal of the human gastrointestinal tract and a frequent cause of bloodstream infections in hospitalized patients. Here, we identify genes that contribute to growth of E. faecium in human serum. We first sequenced the genome of E. faecium E745, a vancomycin-resistant clinical isolate, to completion and then compared its transcriptome during exponential growth in rich medium and in human serum by RNA-seq. This analysis revealed that 27.8% of genes on the E. faecium E745 genome were differentially expressed in these two conditions. A gene cluster with a role in purine biosynthesis was among the most upregulated genes in E. faecium E745 upon growth in serum. A high-throughput transposon sequencing (Tn-seq) approach was used to identify conditionally essential genes in E. faecium E745 during growth in serum. Genes involved in de novo nucleotide biosysnthesis (including pyrK_2, pyrF, purD, purH) and a gene encoding a phosphotransferase system subunit (manY_2) were thus identified to be contributing to E. faecium growth in human serum. Transposon mutants in pyrK_2, pyrF, purD, purH and manY_2 were isolated from the library and their impaired growth in human serum was confirmed. In addition, the pyrK_2 and manY_2 mutants also exhibited significantly attenuated virulence in an intravenous zebrafish infection model. We conclude that genes involved in carbohydrate and nucleotide metabolism of E. faecium are essential for growth in human serum and contribute to the pathogenesis of this organism.