Eosinophils have the capacity to secrete varied cytotoxic proteins. Among the proteins are the eosinophil-associated RNases (EARs): the human eosinophil-derived neurotoxin and eosinophilic cationic protein, and their murine ortholog EARs, which have been shown to be involved in host defense, tissue remodeling, and immunity regulation. However, the signal transduction that regulates EARs secretion in response to physiological stimuli, such as chemokines, has been little studied in human and scarcely in mouse eosinophils, the foremost animal model for eosinophil-associated human diseases.In this study, we aimed to understand the signal transduction involved in the secretion of enzymatically active EARs following chemokine stimulation.Fresh mouse and human eosinophils were stimulated with CCL11 and CCL24, and the secretion of enzymatically active EARs was detected using an RNase activity assay. The involvement of signaling factors or integrins was probed using specific inhibitors and blocking antibodies. Adhesion was evaluated by microscopy.We found that secretion of mouse EARs in response to CCL11 and CCL24 was Gαi -dependent. Both mouse and human eosinophils required the activation of PI3K, ERK, and p38 MAPK. In addition, the adhesion molecules β1 and β2 integrins were found to be crucial for EAR secretion, and we suggest a mechanism in which spreading is obligatory for EAR secretion.Collectively, these data suggest a common CCR3-mediated signaling pathway that leads to EAR secretion in both mouse and human eosinophils. These findings are applicable for eosinophil-mediated host defense and eosinophil-associated diseases.