Neutrophils are the most abundant white blood cells in circulation and are key components of the innate immune response. Clinical and experimental studies support an important role for the neutrophils in a broad spectrum of acute and chronic inflammatory conditions. However, our understanding of nodal points that control neutrophil activation remains incompletely understood. Over the past decade, studies have linked members of the Kruppel-like family of transcription factors (KLFs) to myeloid cell differentiation and function. Here we show that KLF4 is a critical transcriptional regulator of neutrophil biology. KLF4-deficient neutrophils exhibited impaired responses to inflammatory stimulation ex vivo, including reduced production of cytokines and reactive oxygen species, impaired degranulation, and impaired bacterial killing and clearance. Consequently, mice bearing myeloid-specific conditional KLF4 deficiency (K4-cKO) exhibited enhanced susceptibility to bacterial infection but resistance to lipopolysaccharide-induced septic shock and experimental autoimmune encephalomyelitis. Finally, mechanistic studies revealed that the defects in KLF4-deficient neutrophils likely resulted from the defective Toll-like receptor 4-NF-κB signaling. Collectively, these findings identify KLF4 as a novel transcriptional regulator of neutrophil activation.