BACKGROUND: Sacubitril/valsartan (S/V) treatment is beneficial in patients with heart failure with reduced ejection fraction (HFrEF), but its mode of action remains elusive, although it involves the increase in ANP (atrial natriuretic peptide). METHODS: Combining mass spectrometry and enzymatic assay in the plasma of 73 HFrEF patients treated with S/V and controls, we deciphered proANP processing that converts proANP into 4 vasoactive peptides. RESULTS: We found that proANP processing is sequential and involved meprin B, ECE (endothelin-converting enzyme) 1, and ANPEP (aminopeptidase N). This processing is limited in HFrEF patients via the downregulation of proANP production, corin, and meprin B activities by miR-425 and miR1-3p. S/V restored or compensated proANP processing by downregulating miR-425 and miR1-3p, hence increasing levels of proANP-derived bioactive peptides. In contrast, S/V directly and indirectly partially inhibited ECE1 and ANPEP. ECE1 partial inhibition resulted in a lower-than-expected increase in ET1 (endothelin 1), tilting the vasoactive balance toward vasodilation, and possibly hypotension. Furthermore, proANP glycosylation interferes with the midregional proANP assay-a clinical surrogate for proANP production, preventing any pathophysiological interpretation of the results. The analysis of S/V dose escalation with respect to baseline treatments suggests S/V-specific effects. CONCLUSIONS: These findings offer mechanistic evidence to the natriuretic peptide-defective state in HFrEF, which is improved by S/V. These data also strongly suggests that S/V increases plasma ANP by multiple mechanisms that involve 2 microRNAs, besides its protection from NEP (neprilysin) cleavage. Altogether, these data provide new insights on HFrEF pathophysiology and the mode of action of S/V.