Human transthyretin (TTR) is a homotetrameric plasma protein associated with a high percentage of β-sheet, which forms amyloid fibrils and accumulates in tissues or extracellular matrix to cause amyloid diseases. Free energy simulations based on all-atom molecular dynamics simulations were carried out to analyze the effects of the His88 → Arg, Phe, and Tyr mutations on the stability of human TTR. The calculated free energy change differences (ΔΔG) caused by the His → Arg, Phe, and Tyr mutations at position 88 are 6.48 ± 0.45, -9.99 ± 0.54, and 2.66 ± 0.33 kcal/mol, respectively. These calculated free energy change differences between wild type and the mutants are in excellent agreement with prior experimental values. Our simulation results show that the wild type of the TTR is more stable than H88R and H88Y mutants, whereas it is less stable than the H88F mutant. The free energy component analysis shows that the primary contribution to the free energy change difference (ΔΔG) for the His → Arg mutation arises from electrostatic interaction; the ΔΔG for the His → Phe mutation is from van der Waals and electrostatic interactions and that for the His → Tyr mutation from covalent interaction. The simulation results show that the free energy calculation with thermodynamic integration is beneficial for understanding the detailed microscopic mechanism of protein stability. The implications of the results for understanding stabilizing and destabilizing effect of the mutation and the contribution to protein stability are discussed.