Treprostinil, a prostacyclin analogue, is known for alleviating pulmonary arterial hypertension (PAH), a condition characterised by chronic pulmonary vessel obstruction. PAH imposes a significant afterload on the right heart, leading to heart failure. Recent evidence indicated potential direct therapeutic effects of Treprostinil on failing hearts. Treprostinil exerts its actions, in part, by modulating intracellular cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) signalling pathways. This study aimed to explore the multi-level cardiac pharmacology of Treprostinil. Förster resonance energy transfer (FRET) microscopy compared Treprostinil-induced cAMP/PKA responses in cardiomyocytes and cardiac fibroblasts. Gene expression analysis examined prostacyclin receptor dynamics and Treprostinil's impact across the heart. A PAH rat model was utilised to investigate Treprostinil's effects on cAMP/PKA signalling, fibroblasts, and cardiomyocyte contraction. Human atrial fibroblasts were implemented for anti-fibrotic studies under conditions mimicking PAH-related stressors in vitro. Findings indicated that Treprostinil triggered elevated cAMP/PKA responses in the right heart cells compared to the left heart. Furthermore, Treprostinil enhanced right heart cardiomyocyte contraction indirectly through Isoprenaline synergy. In PAH rat models, Treprostinil's efficacy in right ventricular cardiomyocytes was compromised, correlating with reduced cAMP/PKA response. In a murine model, intravenous administration of Treprostinil selectively targeted the right atrium (RA), leading to the upregulation of prostacyclin response genes. Human heart tissue analysis revealed elevated expression of prostacyclin receptors, particularly in RA fibroblasts, corroborated with robust Treprostinil-induced cAMP/PKA responses. PAH models revealed enhanced fibroblast response to Treprostinil. Treprostinil also inhibited human fibroblast activation in vitro. Overall, the study revealed diverse pharmacological actions of Treprostinil within the heart and unravelled cellular targets and mechanisms potentially associated with therapeutic benefit of Treprostinil in modulating cardiac function.