Immune sensing of the Gram-negative bacterial membrane glycolipid lipopolysaccharide (LPS) is both a critical component of host defense against bacterial infection and a contributor to the hyperinflammatory response, potentially leading to sepsis and death. Innate immune activation by LPS is due to the lipid A moiety, an acylated di-glucosamine molecule that can activate inflammatory responses via the extracellular sensor Toll-like receptor 4 (TLR4)/myeloid differentiation 2 (MD2) or the cytosolic sensor caspase-11 (Casp11). The number and length of acyl chains present on bacterial lipid A structures vary across bacterial species and strains, which affects the magnitude of TLR4 and Casp11 activation. TLR4 and Casp11 are thought to respond similarly to various lipid A structures, as tetra-acylated lipid A structures do not activate either sensor, whereas hexa-acylated structures activate both sensors. However, the precise features of lipid A that determine the differential activation of each receptor remain poorly defined, as direct analysis of extracellular and cytosolic responses to the same sources and preparations of LPS/lipid A structures have been limited. To address this question, we used rationally engineered lipid A isolated from a series of bacterial acyl-transferase mutants that produce novel, structurally defined molecules. Intriguingly, we found that the location of specific secondary acyl chains on lipid A resulted in differential recognition by TLR4 or Casp11, providing new insight into the structural features of lipid A required to activate either TLR4 or Casp11. Our findings indicate that TLR4 and Casp11 sense nonoverlapping areas of lipid A chemical space, thereby constraining the ability of Gram-negative pathogens to evade innate immunity.