Objective In homeostatic conditions the peritoneal cavity is populated by resident macrophages. Inflammatory stimuli trigger a phenomenon called macrophage disappearance reaction (MDR), during which resident macrophages become irretrievable from the lavage of the serous cavity. This phenomenon was already observed after different inflammatory insults , but is still incompletely understood. MDR can be associated with cell death, adhesion to neighbouring tissues or migration to the draining lymph nodes or the omentum. MDR is a strategy to face and annihilate the infection by which macrophages, under the control of GATA6, move from the peritoneum to the closest tissues in order to alert the immune system. However, the specific distribution of peritoneal macrophages in MDR is still unclear. In our study, peritoneal macrophages labelled with cell membrane green fluorescent dye DiO were used to study the tracking of peritoneal macrophages in the macrophages disappearance reaction.Methods Peritoneal macrophages labelled with DiO were transplanted to C57BL/6 mice. The macrophage disappearance reaction was induced by lipopolysaccharide (LPS) in vivo. Fluorescence microscope and flow cytometry were used to detect the number and fluorescence intensity of DiO-labelled peritoneal macrophages. The tissues of mice were separated and collected, and frozen sections were made to detect the distribution of DiO-labelled peritoneal macrophages.Results The observation by fluorescence microscope and flow cytometry showed that intraperitoneal injection of LPS could significantly reduce the number and fluorescence intensity of DiO-labelled peritoneal macrophages. Peritoneal macrophages that disappeared during the macrophage disappearance reaction were found distributed in the liver, thymus and spleen by frozen sections. DiO labelling peritoneal macrophages does not affect cell viability and with long-term stability in vivo, indicating that DiO may be a safe and effective green fluorescent dye for tracking the distribution of peritoneal macrophages.Conclusion This research method will provide a convenient and effective experimental means for exploring the dynamic changes and related biological phenomena of peritoneal macrophages during MDR. Furthermore, it laid a foundation for further research on the causes and mechanisms of MDR.