Background and Purpose Upon stimulation, neutrophils release their nuclear contents called neutrophil extracellular traps (NETs), which contain unfolded chromatin and lysosomal enzymes. NETs have been demonstrated to play a critical role in host defence, although the role of PGE2, a bioactive substance generated in inflammatory tissues, in the formation of NETs remains unclear. Experimental Approach The effects of PGE2, agonists and antagonists of its receptors, and modulators of the cAMP-PKA pathway on the formation of NETs were examined in vitro in isolated neutrophils and in vivo in a newly established mouse model. Key Results PGE2 inhibited PMA-induced NET formation in vitro through EP2 and EP4 Gαs-coupled receptors. Incubation with a cell-permeable cAMP analogue, dibutyryl cAMP, or various inhibitors of a cAMP-degrading enzyme, PDE, also suppressed NET formation. In the assay established here, where an agarose gel was s.c. implanted in mice and NET formation was detected on the surface of the gel, the extent of the NET formed was inhibited in agarose gels containing rolipram, a PDE4 inhibitor, and butaprost, an EP2 receptor agonist. Conclusions and Implications PGE2 inhibits NET formation through the production of cAMP. These findings will contribute to the development of novel treatments for NETosis-related diseases.