Neutrophil chemotaxis to sites of inflammation is a critical process during normal immune responses to tissue injury and infection and pathological immune responses leading to chronic inflammation. Although progress has been made in understanding the mechanisms that promote neutrophil recruitment to inflamed tissue, the mechanisms that regulate the resolution phase of the inflammatory response have remained relatively elusive. To define the mechanisms that regulate neutrophil-mediated inflammation in vivo, we have developed a novel transgenic zebrafish in which the neutrophils express GFP under control of the myeloperoxidase promoter (zMPO:GFP). Tissue injury induces a robust, inflammatory response, which is characterized by the rapid chemotaxis of neutrophils to the wound site. In vivo time-lapse imaging shows that neutrophils subsequently display directed retrograde chemotaxis back toward the vasculature. These findings implicate retrograde chemotaxis as a novel mechanism that regulates the resolution phase of the inflammatory response. The zMPO:GFP zebrafish provides unique insight into the mechanisms of neutrophil-mediated inflammation and thereby offers opportunities to identify new regulators of the inflammatory response in vivo.