Abstract Mouse models often serve as a selective gate for drugs developed to treat patients with inflammation and sepsis. However, mice are much more resistant than humans to intravenous challenge with LPS, a cell wall component of Gram-negative bacteria. It is common to detect up to 106 CFU/ml of live bacteria in the blood in many mouse models of bacterial challenge but less than 100 CFU/ml of bacteria in the blood of human adults with Gram-negative sepsis. These substantial differences between mice and humans during infections are unexplained, unstudied and usually ignored. We studied and compared the growth of four encapsulated, clinically relevant strains of Escherichia coli (O4:K54:H5, O16:K1:H6, O18:K1:H7, O25:K5:H1) and Pseudomonas aeruginosa 12.4.4 in fresh human and mouse blood at 37°C for 30, 90 and 180 minutes. We also measured TNF and IL-6 levels in the supernatants, and performed a phagocytosis assay by using fluorescein isothiocyanate-labeled heat-killed bacteria incubated with human and mouse blood followed by flow cytometry. We found that E. coli and P. aeruginosa multiplied in mouse blood and plasma, but that the colony counts of each were decreased in human blood and plasma. More E. coli and P. aeruginosa were phagocytosed by leukocytes in human than those in mouse blood, but this difference reflected larger numbers of neutrophils in human blood than in mouse blood. Large amounts of pro-inflammatory cytokines TNF and IL-6 were detected in human but not in mouse blood after live bacterial inoculation. In conclusion, our results suggest that bacterial killing, phagocytosis, and cytokine induction in the blood compartment of patients with Gram-negative bacteremia may not be well mimicked in mouse models of bacterial challenge.