Rationale: Our understanding of the pathophysiology of sepsis remains incomplete. Genome-wide study offers an unbiased, system biology approach to examine the expression patterns of circulating leukocytes and may reveal novel insights into the host response to sepsis. Objectives: We examined whether gene-expression profiling of neutrophils could identify signature genes and important pathways in the clinical syndrome of sepsis. Methods: Gene-expression profiling was performed using oligonucleotide microarrays on peripheral blood samples of 94 critically ill patients (71 were septic and 23 non-septic). Using a supervised learning algorithm based on support vector machine, a molecular signature of sepsis was generated from a training set of 44 samples and validated in an independent set of 50 samples. The diagnostic performance of the signature genes was assessed against a reference standard based on the International Sepsis Forum Consensus Conference definition of infection. Measurements and Main Results: A set of 50 signature genes correctly identified sepsis with a prediction accuracy of 91% and 88% in the training and validation sets respectively. The diagnostic performance remained high regardless of patient's age, co-morbidities or prior antibiotic treatment. Compared to control, genes involved in immune modulation and inflammatory response had reduced expression in patients with sepsis. In particular, the activation of nuclear factor-B pathway was reduced while its inhibitor gene NFBIA was significantly up-regulated. Conclusion: The signature genes reflect suppression of neutrophils' immune and inflammatory function by sepsis. Gene expression profiling therefore provides a novel approach to advance our understanding of the host response in sepsis.