Modification of a lipid A moiety in Gram-negative bacterial LPS to a less acylated form is thought to facilitate bacterial evasion of host innate immunity, thereby enhancing pathogenicity. The contribution of less-acylated lipid A to interactions of whole bacterial cells with host cells (especially in humans) remains unclear. Mutant strains of Salmonella enterica serovar Typhimurium with fewer acylated groups were generated. The major lipid A form in wild-type (WT) and the mutant KCS237 strain is hexa-acylated; in mutant strains KCS311 and KCS324 it is penta-acylated; and in KCS369 it is tetra-acylated. WT and KCS237 formalin-killed and live bacteria, as well as their LPS, strongly stimulated production of pro-inflammatory cytokines in human U937 cells; this stimulation was suppressed by TLR4 suppressors. LPS of other mutants produced no agonistic activity, but strong antagonistic activity, while their formalin-killed and live bacteria preparations had weak agonistic and no antagonistic activity. Moreover, these less-acylated mutants had increased resistance to phagocytosis by U937 cells. Our results indicate that a decrease of one acyl group (from six to five) is enough to allow Salmonella to evade human innate immunity and that the antagonistic activity of less-acylated lipid A is not utilized for this evasion.