Thiamin pyrophosphokinase catalyzes the pyrophosphorylation of thiamin to thiamin pyrophosphate in the presence of ATP and Mgtt The kinetic properties of human thiamin pyrophosphokinase (hTPK1) were investigated using purified histidine-tagged recombinant protein. The plots of the initial velocity against MgATP concentrations gave a sigmoidal character when Mg²⁺/ATP was maintained at 1. However, the addition of an excess amount of Mg²⁺ resulted in the restoration of activity at lower concentrations of MgATP A steady-state kinetics study led us to conclude that the kinase reaction obeys a ping-ong mechanism. Site-directed mutagenesis was also performed on hTPK1 to examine the contributions of eight strictly conserved residues in thiamin pyrophosphokinase on the kinetic properties. Mutations D71N, D73N, and D100N reduced k_cat markedly, indicating that these aspartic acids play a crucial role in carrying out the catalytic process of hTPK1. A selective decrease in the k_cat/Km^thiamin value was observed in the D133N mutant, whereas the k_cat/Km^ATV values of T99A and R131G were significantly decreased. Interestingly, the replacement of Gln-96 with Glu caused an increase in the k_cat/Km^thiamin value (3.53-fold of the wild-type). It was therefore suggested that the residues Gln-96, Thr-99, Arg-131, and Asp-133 are conserved as functionally significant components for substrate recognition in thiamin pyrophosphokinase.