A plethora of studies have suggested the involvement of various eicosanoids in heart failure. The aim of this study was to profile eicosanoid release from isolated murine heart at transition and end-stage phases of heart failure (HF) in Tgαq*44 mice as compared with age-matched wild-type FVB mice. Using an UPLC-MS/MS-based method, the concentration of selected eicosanoids was measured in cardiac effluents collected from isolated perfused mice hearts according to the Langendorff technique in Tgαq*44 and FVB mice (8- and 12-month-old) in basal conditions and in response to bolus injection of arachidonic acid (AA), a major substrate for eicosanoids. In basal conditions, only some eicosanoids were detected in the coronary effluents, with 6-keto-PGF1α, PGD2, 12-HETE detected at the highest concentration. In response to AA, a wide range of its metabolites was detected, including not only prostanoids and HETEs, but also EETs and DHETs. Cardiac production of 6-keto-PGF1α, PGD2, PGE2, PGF2α, TXB2 in basal conditions was unchanged at the transition phase of HF, whereas it was increased at the end-stage of disease in Tgαq*44 mice as compared with age-matched FVB mice. In response to AA, the synthesis of PGE2, 12-, 15-HETEs, 8,9-, 11,12-DHETs were also elevated at the end-stage phase of HF in Tgαq*44 mice as compared to healthy animals. AA-induced vasodilation effect was greater at the end-stage phase of HF in Tgαq*44 mice as compared with age-matched FVB mice, but it was not changed at the transition phase of the disease. In conclusion, eicosanoid profiling in isolated perfused heart pointed to PGI2, PGD2 and 12-HETE as the most abundant AA metabolites of the isolated murine heart. In Tgαq*44 mice, the end-stage phase of heart failure was accompanied by major activation of cyclooxygenase pathways (PGI2, TXA2, PGD2, PGE2, PGF2α), 8,9-, 11,12-EET/DHETs pathways and 12-, 15-HETEs pathways in the heart.