Proliferation of bacterial pathogens in blood represents one of the most dangerous stages of infection. Growth in blood serum depends on the ability of a pathogen to adjust metabolism to match the availability of nutrients. Although certain nutrients are scarce in blood and need to be de novo synthesized by proliferating bacteria, it is unclear which metabolic pathways are critical for bacterial growth in blood. In this study, we identified metabolic functions that are essential specifically for bacterial growth in the bloodstream. We used two principally different but complementing techniques to comprehensively identify genes that are required for the growth of Escherichia coli in human serum. A microarray-based and a dye-based mutant screening approach were independently used to screen a library of 3,985 single-gene deletion mutants in all non-essential genes of E. coli (Keio collection). A majority of the mutants identified consistently by both approaches carried a deletion of a gene involved in either the purine or pyrimidine nucleotide biosynthetic pathway and showed a 20- to 1,000-fold drop in viable cell counts as compared to wild-type E. coli after 24 h of growth in human serum. This suggests that the scarcity of nucleotide precursors, but not other nutrients, is the key limitation for bacterial growth in serum. Inactivation of nucleotide biosynthesis genes in another gram-negative pathogen, Salmonella enterica, and in the gram-positive pathogen Bacillus anthracis, prevented their growth in human serum. The growth of the mutants could be rescued by genetic complementation or by addition of appropriate nucleotide bases to human serum. Furthermore, the virulence of the B. anthracis purE mutant, defective in purine biosynthesis, was dramatically attenuated in a murine model of bacteremia. Our data indicate that de novo nucleotide biosynthesis represents the single most critical metabolic function for bacterial growth in blood and reveal the corresponding enzymes as putative antibiotic targets for the treatment of bloodstream infections.