AbstractAs an important part of tumor microenvironment, neutrophils are poorly understood due to their spatiotemporal heterogeneity in tumorigenesis. Here we defined, at single-cell resolution, CD44−CXCR2−neutrophils as tumor-specific neutrophils (tsNeus) in both mouse and human gastric cancer (GC). We uncovered a Hippo regulon in neutrophils with unique YAP signature genes (e.g.,ICAM1,CD14,EGR1) distinct from those identified in epithelial and/or cancer cells. Importantly, knockout of YAP/TAZ in neutrophils impaired their differentiation into CD54+tsNeus and reduced their antitumor activity, leading to accelerated GC progression. Moreover, the relative amounts of CD54+tsNeus were found to be negatively associated with GC progression and positively associated with patient survival. Interestingly, GC patients receiving neoadjuvant chemotherapy had increased numbers of CD54+tsNeus. Furthermore, pharmacologically enhancing YAP activity selectively activated neutrophils to suppress refractory GC, with no significant inflammation-related side effects. Thus, our work characterized tumor-specific neutrophils in GC and revealed an essential role of YAP/TAZ-CD54 axis in tsNeus, opening a new possibility to develop neutrophil-based antitumor therapeutics.