is a Gram-positive bacterium and zoonotic pathogen that causes meningitis and sepsis in pigs and humans. The aim of this study was to identify genes required for infection. We created Tn-Seq libraries in a virulent strain 10, which was used to inoculate pigs in an intrathecal experimental infection. Comparative analysis of the relative abundance of mutants recovered from different sites of infection (blood, cerebrospinal fluid, and meninges of the brain) identified 361 conditionally essential genes, i.e. required for infection, which is about 18% of the genome. The conditionally essential genes were primarily involved in metabolic and transport processes, regulation, ribosomal structure and biogenesis, transcription, and cell wall membrane and envelope biogenesis, stress defenses, and immune evasion. Directed mutants were created in a set of 10 genes of different genetic ontologies and their role was determined in models. Mutants showed different levels of sensitivity to survival in whole blood, serum, cerebrospinal fluid, thermic shock, and stress conditions, as compared to the wild type. Additionally, the role of three selected mutants was validated in co-infection experiments in which pigs were infected with both wild type and isogenic mutant strains. The genetic determinants of infection identified in this work contribute to novel insights in pathogenesis and could serve as targets for novel vaccines or antimicrobial drugs.