Phosphorylation of the voltage-gated Na+ (Na-V) channel Na(V)1.5 regulates cardiac excitability, yet the phosphorylation sites regulating its function and the underlying mechanisms remain largely unknown. Using a systematic, quantitative phosphoproteomic approach, we analyzed Na(V)1.5 channel complexes purified from nonfailing and failing mouse left ventricles, and we identified 42 phosphorylation sites on Na(V)1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic Na(V)1.5 mutants revealed the roles of three phosphosites in regulating Na(V)1.5 channel expression and gating. The phosphorylated serines S664 and S667 regulate the voltage dependence of channel activation in a cumulative manner, whereas the nearby S671, the phosphorylation of which is increased in failing hearts, regulates cell surface Na(V)1.5 expression and peak Na' current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, our results demonstrate that ventricular Na(V)1.5 is highly phosphorylated and that the phosphorylation-dependent regulation of Na(V)1.5 channels is highly complex, site specific, and dynamic.