The pathogenic bacterium Neisseria meningitidis is an important cause of septicemia and meningitis, especially in childhood. The establishment and maintenance of bacteremic infection is a pre-requisite for all the pathological sequelae of meningococcal infection. To further understand the genetic basis of this essential step in pathogenesis, we analyzed a library of 2,850 insertional mutants of N. meningitidis for their capacity to cause systemic infection in an infant rat model. The library was constructed by in vitro modification of Neisseria genomic DNA with the purified components of Tn10 transposition. We identified 73 genes in the N. meningitidis genome that are essential for bacteremic disease. Eight insertions were in genes encoding known pathogenicity factors. Involvement of the remaining 65 genes in meningocoocal pathogenesis has not been demonstrated previously, and the identification of these genes provides insights into the pathogenic mechanisms that underlie meningococcal infection. Our results provide a genome-wide analysis of the attributes of N. meningitidis required for disseminated infection, and may lead to new interventions to prevent and treat meningococcal infection.