Phosphorylation of the cardiac myosin regulatory light chain (cMLC2v) modulates myosin's function and its interaction with actin, thus able to modulate contraction. While small rodents carry two phosphorylation sites (S14, S15), the human cMLC2v only has a single phosphorylation site (S15). In humans, S14 is replaced with neutral Asparagine (N14) that can undergo deamidation to form Aspartate (Asp, D, a charged residue) which has been observed in heart failure. However, the impact of N14D transformation on S15 phosphorylation is unknown. Consequently, we expressed human recombinant wild type (Wt), mutant S15A and N14D/S15 cMLC2v proteins and phosphorylated with relevant kinases in vitro. Following, proteins were analyzed via 1D and 2D SDS-PAGE. Staining 1D gels with Pro-Q diamond, showed Wt cMLC2v was phosphorylated by all 4 kinases used, while the S15A mutant served as a negative control. Interestingly, N14D/S15 cMLC2v also showed no indication of phosphorylation. To investigate further, 2D-Western blots were probed with a phospho-MYL2 S15 antibody. Phosphorylation of Wt cMLC2v revealed spots positive for phospho-S15 compared to unphosphorylated Wt cMLC2v. 2D-PAGE analysis of the N14D/S15 and S15A cMLC2v phospho-species was negative with kinase treatment, validating our initial results. Correlating these data with patient biopsies, blinded analysis demonstrated a decrease in cMLC2v phosphorylation occurs in patients with heart failure. While the influence of N14D transformation on the modulation of contraction remains unknown, these data indicate deamidation of Asn 14 blocks access to a critical regulatory phosphorylation motif (S15) in human cMLC2v. Thus, N14D transformation may play a role in the diminished levels of cMLC2v phosphorylation reported in heart failure.