Macrophage extracellular traps (METs) represent a recently discovered complex defence mechanism that is distinct from phagocytosis and involves the release of DNA and antibacterial proteins. They play an important role in pathogen removal, and calcium ions (Ca(2+)) have also been reported to be involved. In the present study, we identified METotic cells using digitonin as an alternative to Triton X-100, coupled with immunofluorescence staining using lamin antibodies. The limited permeability of digitonin ensures exclusive intranuclear antibody labelling of MET cells, therefore providing a straightforward and intuitive differentiation method. We found that under lipopolysaccharide stimulation, macrophages undergo store-operated Ca(2+) entry (SOCE) to facilitate Ca(2+) influx. Elevation of cytoplasmic Ca(2+) levels by SOCE promotes the generation of superoxide anions by NADPH oxidase (NOX), ultimately leading to METosis. In summary, our study strengthens the role of Ca(2+) in NOX-dependent METosis, which differs from previous studies focusing on Ca(2+) in the NOX-independent pathway. Our research reveals that Ca(2+)-mediated regulation of NOX plays a crucial role in METosis, especially in SOCE, and provides novel ideas for future research.