Brucella abortus is a class III zoonotic bacterial pathogen able to survive and replicate inside host cells, including macrophages. Here we report a multidimensional transposon sequencing analysis to identify genes essential for Brucella abortus growth in rich medium and replication in RAW 264.7 macrophages. The construction of a dense transposon mutant library and mapping of 929,769 unique mini-Tn5 insertion sites in the genome allowed identification of 491 essential coding sequences and essential segments in the B. abortus genome. Chromosome II carries a lower proportion (5%) of essential genes than chromosome I (19%), supporting the hypothesis of a recent acquisition of a megaplasmid as the origin of chromosome II. Temporally resolved transposon sequencing analysis as a function of macrophage infection stages identified 79 genes with a specific attenuation phenotype in macrophages, at either 2, 5, or 24 h postinfection, and 86 genes for which the attenuated mutant phenotype correlated with a growth defect on plates. We identified 48 genes required for intracellular growth, including the virB operon, encoding the type IV secretion system, which supports the validity of the screen. The remaining genes encode amino acid and pyrimidine biosynthesis, electron transfer systems, transcriptional regulators, and transporters. In particular, we report the need of an intact pyrimidine nucleotide biosynthesis pathway in order for B. abortus to proliferate inside RAW 264.7 macrophages.