E5564, a structural analog of the lipid A portion of lipopolysaccharide (LPS), is a potent antagonist of the biochemical and physiologic effects of LPS in several in vitro and in vivo models and is currently under clinical development as a possible therapeutic for the treatment of sepsis and septic shock. The objectives of this study were to (1) assess the safety and tolerability of E5564 following a 30‐minute intravenous (IV) infusion, (2) evaluate the pharmacokinetic profile of E5564, and (3) measure the ability of E5564 to block LPS stimulation ex vivo in blood taken from subjects up to 8 hours after ending the infusion. Healthy male volunteers (n= 7/dose group) were randomly assigned to each of four dose levels (350, 1000, 2000, or 3500 μg). Within each dose group, 5 subjects received drug and 2 received placebo. E5564 or matching placebo was administered by a 30‐minute infusion, and blood samples were collected at predetermined time points. All doses of E5564 were demonstrated to be safe and well tolerated. E5564 plasma concentrations were determined using a validated LC/MS/MS method. The C_max and AUC of E5564 increased in a dose‐proportional manner. E5564 pharma‐ cokinetics were characterized by a slow clearance (0.67–0.95 mL/h/kg), a small volume of distribution (41–54 mL/kg), and a relatively long elimination half‐life (42–51 h). As measured in the ex vivo assay, E5564 inhibited LPS‐induced tumor necrosis factor–α (TNF‐α) in a dose‐dependent manner, and at the higher doses (2 and 3.5 mg), antagonistic activity was measurable up to 8 hours postinfusion. E5564 lacked LPS‐like agonist activity at doses up to 3.5 mg. Taken together, we believe that E5564 is a safe, potent antagonist of LPS in blood and will likely benefit patients in the treatment of LPS‐related diseases.