Both cardiac and skeletal calsequestrin (CASQ2 and CASQ1) serve as a major Ca(2+) storage/buffer protein in the sarcoplasmic reticulum (SR) by sequestering and releasing large numbers of Ca(2+) ions during each muscular contraction and relaxation cycle. CASQ isolated from various species often exists in a phosphorylated form, but phosphorylation's role is not yet understood. Here, the authors identified two phosphorylation sites, Ser(385) and Ser(393), for the first time, in human CASQ2 (hCASQ2) by mass-spectroscopy and evaluated the consequences of such phosphorylation. Substitution of these two serines with phosphoserine-mimicking aspartic-acid residues results in a significant increase in helical content, solubility and Ca(2+)-binding capacity above 6 mM [Ca(2+)]. However, neither substitution of Ser(385) nor Ser(393) alone produce any significant changes. Based on the crystal structures of hCASQ2, Ca(2+) binding capacity data, turbidity, and light scattering profiles, it was propose that phosphorylation at these two positions produces a disorder-to-order or coil-to-helix transition of the C-terminus, which in turn provides a more stable network of polyanions. Therefore, considering all the previous reports and the new data, the observed dynamic in vivo phosphorylation of CASQ could provide the basis not only for effective regulation of Ca(2+) buffering capacity, but also for the junctional SR trafficking mechanism.