In the heart, cAMP is a key regulator of excitation—contraction coupling and its biological effects are mainly associated with the activity of protein kinase A (PKA). The aim of this study was to investigate the contribution of the cAMP-binding protein Epac (Exchange protein directly activated by cAMP) in the regulation of the contractile properties of rat ventricular cardiac myocytes. We report that both PKA and Epac increased cardiac sarcomere contraction but through opposite mechanisms. Differently from PKA, selective Epac activation by the cAMP analog 8-pCPT reduced Ca2+ transient amplitude and increased cell shortening in intact cardiomyocytes as well as myofilament Ca2+ sensitivity in permeabilized cardiomyocytes. Moreover, ventricular myocytes, which were infected in vivo with a constitutively active form of Epac, showed enhanced myofilament Ca2+ sensitivity compared to control cells infected with GFP alone. At the molecular level, Epac increased phosphorylation of two key sarcomeric proteins, cardiac Troponin I (cTnI) and cardiac Myosin Binding Protein-C (cMyBP-C). The effects of Epac activation on myofilament Ca2+ sensitivity and on cTnI and cMyBP-C phosphorylation were independent of PKA, and were blocked by protein kinase C (PKC) and Ca2+ calmodulin kinase II (CaMKII) inhibitors. Altogether these findings identify Epac as a new regulator of myofilament function.