The cardiac sodium–calcium exchanger (NCX1) is important for normal Na+ and Ca2+ homeostasis and cardiomyocyte relaxation and contraction. NCX1 activity is reduced by its endogenous inhibitor phosphorylated phospholemman (pSer68-PLM). Disrupting the pSer68-PLM-NCX1 interaction is therefore a potential therapeutic strategy to reverse NCX1 inhibition in cardiac disease. We have previously designed a high affinity NCX1-PLM disruptor peptide (OPT) that reverses the inhibitory pSer68-PLM-NCX1 interaction in HEK293 cells. In the present study, we performed N and C-terminal truncation analyses of OPT and identified PYKEIEQLIELANYQV as the minimal pSer68-PLM binding sequence. To increase peptide stability in human serum, we replaced the proline in its N-terminus with an N-methyl-proline (NOPT) after identification of N-terminus as substitution tolerant by two dimensional peptide array analysis. Mass spectrometry analysis revealed that the half-life of the novel NOPT peptide was increased 10-fold compared to that of OPT. Biotinylated NOPT pulled down endogenous PLM from rat left ventricle lysate and exhibited pSer68-PLM binding in an ELISA-based assay. Importantly, NOPT reduced the PLM-NCX1 interaction in an ELISA-based assay and cell permeable NOPT-TAT increased the NCX1 activity in adult cardiomyocytes isolated from both wild type (WT) and HF animals. Finally, we found that NOPT interacted with pSer68-PLM with a KD value of 2.7±3.8 μM, whereas the NCX1 cytoplasmic part interacted with PLM and pSer68-PLM with KD values of 14±2 nM and 24±14 nM. In conclusion, we have here developed a proteolytically stable NCX1-PLM disruptor peptide that upregulates NCX1 activity in WT and HF cardiomyocytes.