Since rapidly evolving pathogens exert intense pressure on the immune system to co-evolve, substantial differences in innate immune function between humans and mice are likely, but the extent and mechanisms of such differences have not been systematically addressed. We investigated innate immune diversification by analysing inter-species differences in the transcriptional responses of primary human and mouse macrophages to the TLR4 agonist, bacterial lipopolysaccharide (LPS). Using a custom platform permitting cross-species interrogation coupled with deep sequencing of mRNA 5’ ends, we identified extensive divergence in LPS-regulated orthologous gene expression between human and mouse macrophages (24% of orthologs, http://www.macgate.qfab.org). We further demonstrate concordant regulation of human-specific LPS target genes in primary pig macrophages. Divergently regulated (DR) orthologs were enriched for genes encoding cellular “inputs” such as cell surface receptors (e.g. TLR6, IL-7Ra), and functional “outputs” such as inflammatory cytokines/chemokines (e.g. CCL20, CXCL13). Conversely, intracellular signaling components linking inputs to outputs were typically concordantly regulated. Functional consequences of divergent gene regulation were confirmed by showing LPS pre-treatment boosts subsequent TLR6 responses in mouse but not human macrophages, in keeping with mouse-specific TLR6 induction. DR genes were associated with a large dynamic range of gene expression, and specific promoter architectural features (TATA box enrichment, CpG island depletion). Surprisingly, regulatory divergence was also associated with enhanced inter-species promoter conservation. Thus, the genes controlled by complex, highly conserved promoters that facilitate dynamic regulation are also the most susceptible to evolutionary change. Our study maps the strengths and weaknesses of the mouse model and identifies novel human-specific responses as therapeutic targets for infectious and inflammatory diseases (Schroder et al., PNAS, 2012).