Effect of mutations mimicking phosphorylation on the structure of human 14-3-3zeta protein was analyzed by different methods. Mutation S58E increased intrinsic Trp fluorescence and binding of bis-ANS to 14-3-3. At low protein concentration mutation S58E increased the probability of dissociation of dimeric 14-3-3 and its susceptibility to proteolysis. Mutation S184E slightly increased Stokes radius and thermal stability of 14-3-3. Mutation T232E induced only small increase of Stokes radius and sedimentation coefficient that probably reflect the changes in the size or shape of 14-3-3. At low protein concentration the triple mutant S58E/S184E/T232E tended to dissociate, whereas at high concentration its properties were comparable with those of the wild type protein. The triple mutant was highly susceptible to proteolysis. Thus, mutation mimicking phosphorylation of Ser58 destabilized, whereas mutation of Ser184 induced stabilization of 14-3-3zeta structure.