Neutrophils are crucial for immunity and play important roles in inflammatory diseases; however, mouse models selectively deficient in neutrophils are limited, and neutrophil-specific diphtheria toxin (DT)-based depletion system has not yet been established. In this study, we generated a novel knock-in mouse model expressing diphtheria toxin receptor (DTR) under control of the endogenous Ly6G promoter. We showed that DTR expression was restricted to Ly6G neutrophils and complete depletion of neutrophils could be achieved by DT treatment at 24-48 h intervals. We characterized the effects of specific neutrophil depletion in mice at steady-state, with acute inflammation and during tumor growth. Our study presents a valuable new tool to study the roles of neutrophils in the immune system and during tumor progression.