P2X2 receptor (P2X2R) belongs to the family of purinergic receptors that have been shown to play important roles in regulating host innate immune response. Although the immunologic significance of P2X2R has been studied in mammals, the presence and immune relevance of P2X2R in fish remains unclear. In this study we extended our previous observations by identifying and characterizing a P2X2R ortholog (termed PoP2X2R) from Japanese flounder (Paralichthys olivaceus). Quantitative real-time PCR analysis revealed that PoP2X2R mRNA transcripts are widely distributed in all examined normal tissues and are dominantly expressed in hepatopancreas tissue. In addition, we for the first time showed that multiple P2XR subtypes, including P2X2R, P2X4R and P2X7R are co-expressed in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood lymphocytes (PBLs), indicating that they may assemble into hetero-receptor complex or interact in the form of homotrimers to trigger diverse purinergic signaling in the Japanese flounder immune cells. Compared with the known Japanese flounder P2X4 and P2X7 receptors, however, PoP2X2R is much more abundantly expressed in the Japanese flounder HKM cells, suggesting that PoP2X2R may play an important role in this type of immune cells. Glycosylation and immunohistochemistry analyses revealed that PoP2X2R is a glycoprotein expressed on the plasma membrane. Immune challenges experiments showed that PoP2X2R was significantly induced by LPS, poly(I:C) and zymosan stimulations in the HKM and PBL cells, and by Edwardsiella tarda infections in spleen and gill tissues as well. Taken together, we have identified and characterized a new P2X2R member that is involved in fish innate immune response.