Prostaglandin D2 (PGD2) may act against myocardial ischemia–reperfusion (I/R) injury and play an anti-inflammatory role in the heart. Although the effect of PGD2 in regulation of ANP secretion of the atrium was reported, the mechanisms involved are not clearly identified. The aim of the present study was to investigate whether PGD2 can regulate ANP secretion in the isolated perfused beating rat atrium, and its underlying mechanisms. PGD2 (0.1 to 10 μM) significantly increased atrial ANP secretion concomitantly with positive inotropy in a dosedependent manner. Effects of PGD2 on atrial ANP secretion and mechanical dynamics were abolished by AH-6809 (1.0 μM) and AL-8810 (1.0 μM), PGD2 and prostaglandin F2α (PGF2α) receptor antagonists, respectively. Moreover, PGD2 clearly upregulated atrial peroxisome proliferator-activated receptor gamma (PPARγ) and the PGD2 metabolite 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2, 0.1 μM) dramatically increased atrial ANP secretion. Increased ANP secretions induced by PGD2 and 15d-PGJ2 were completely blocked by the PPARγ antagonist GW9662 (0.1 μM). PD98059 (10.0 μM) and LY294002 (1.0 μM), antagonists of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling, respectively, significantly attenuated the increase of atrial ANP secretion by PGD2. These results indicated that PGD2 stimulated atrial ANP secretion and promoted positive inotropy by activating PPARγ in beating rat atria. MAPK/ERK and PI3K/Akt signaling pathways were each partially involved in regulating PGD2-induced atrial ANP secretion.