Neuromuscular junctions (NMJs) are specialized, vital interfaces between nervous system and muscle. Their formation is characterized by high density clustering of the neurotransmitter receptor acetylcholine receptor (AChR). The central regulator of AChR clustering and thus ontogenesis of the postsynaptic density is the receptor tyrosine kinase Muscle Specific Kinase (MuSK). Mice lacking MuSK die perinatally and dysregulation of MuSK causes a heterogeneous group of myasthenic diseases. MuSK activation is controlled by multiple autoinhibitory mechanisms: extracellular ligand binding facilitates oligomerization of MuSK via its extracellular receptor region, leading to intracellular adaptor protein binding and autophosphorylation in its tyrosine kinase domain (TKD). We characterized a novel serine phosphorylation site (S751) whose phosphorylation is implicated to modulate MuSK activation during NMJ maintenance. We expressed the TKD of recombinant human MuSK and engineered a phosphorylation mimicking mutant equivalent to murine S751D. We used comparative enzyme kinetic measurements, x-ray crystallography and hydrogen deuterium exchange mass spectrometry and show that S751D increased substrate affinity of the kinase by breaking inhibitory hydrogen bonds and -stacking interactions, resulting in overall increased flexibility of the TKD. The overall increased flexibility did not affect rigidity of sidechains whose mutation causes congenital myasthenic syndromes, supporting that S751 phosphorylation did not interfere with protein folding or stability. These data suggest that S751 phosphorylation primes MuSK for activation. Radiometric kinase profiling experiments suggested that the peptide surrounding S751 is a substrate for Ca2+/calmodulin dependent protein kinase II beta (CaMK2β). We validated this interaction in heterologous cells and found that Crispr/Cas9 mediated ablation of CaMK2β in cultured muscle cells perturbed AChR clustering but not MuSK phosphorylation. Subsequent histological characterization of NMJs of ataxic mice that lack all CaMK2β splice variants, or an established mouse model for myotonic dystrophy type 1 that lacks the muscle specific splice variant CaMK2βM specifically, indicated protective isozyme compensation. Taken together, our detailed characterization indicates a novel kinase regulatory mechanism that links NMJ formation or maintenance to calcium signaling.