14-3-3 proteins are a family of highly conserved dimeric proteins that bind and regulate the functions of different phosphoproteins and some non-phosphoproteins. While specific ligand proteins of 14-3-3 have been identified and their associated functions have been described, the exact mechanism of ligand association/dissociation which is a key determinant of temporal regulation of 14-3-3 function remains largely unknown. These proteins have also been credited with enzymatic activities. However, the mechanism and significance of enzymatic function have not been well established. We had earlier reported that many human 14-3-3 isoforms but not 14-3-3s can hydrolyze ATP. Mutations that enhance or decrease the activity were identified and two binding pockets were proposed. However, the catalytic residues involved and the significance of this enzymatic function remained unclear. Here, guided by a new algorithm, we confirm that the peptide binding pocket of 14-3-3 is also the ATPase active site. Two glutamic acid residues are involved in the hydrolysis of ATP, probably aided by bound water. Using limited proteolysis coupled to mass spectrometry, we confirm the above results and substantiate the second ATP binding pocket at the dimer interface. We navigated through the various peptide ligands of 14-3-3 to test the effect of ATP binding on ligand association/dissociation. We found that the binding of one of the non-phosphopeptides was significantly affected by the presence of ATP and ATPγS but such an effect was not seen in any tested phosphopeptides. In depth, experiments suggest that both the ATP hydrolysis and non-phosphopeptide binding require binding of ATP/ATPγS at the dimer interface. These studies unveil the hidden allosteric properties of the 14-3-3 proteins and their role in excluding specific ligands from the binding pocket. Such selectivity may play an important context-specific regulation of 14-3-3 functions. We hereby propose that 14-3-3 is an unconventional ATPase lacking classical ATP binding motifs and folds, but executes catalysis via a well-known catalytic mechanism.