Both cardiac and skeletal Calsequestrin (CSQ2 and CSQ1) buffer the calcium concentration in sarcoplasmic reticulum (SR) during the excitation-contraction coupling and regulate the Ryanodine receptor (RyR) activity through the interaction with Junctin (JNT) /Triadin (TRN). CSQ2 contains a longer C terminal tail which possesses two conserved serine compared with CSQ1. To identify the interaction region of JNT which is responsible for CSQ2 interaction, JNT was constructed with different length and fused to GST protein for GST pull-down assay. The third KEKE motif of JNT was identified as the region of JNT for CSQ2 recognition. Unlike the interaction of TRN and CSQ2 in which the deletion of CSQ2 C terminal abolished the interaction between the two proteins, for JNT and CSQ2 interaction, the C terminal of CSQ2 is important but not the only region for JNT binding. Deletion of CSQ2 C terminal only reduced its interaction with JNT. Furthermore, phosphorylation of the serine residues on the C terminal of CSQ2 enhanced its interaction with JNT.