In eukaryotes, there are still steps of the vitamin B1 biosynthetic pathway not completely understood. In Arabidopsis thaliana, THI1 protein has been associated with the synthesis of the thiazole ring, a finding supported by the identification of a thiamine pyrophosphate (TPP)-like compound in its structure. Here, we investigated THI1 and its mutant THI1(A140V), responsible for the thiamin auxotrophy in a A. thaliana mutant line, aiming to clarify the impact of this mutation in the stability and activity of THI1. Recently, the THI1 orthologue (THI4) was revealed to be responsible for the donation of the sulfur atom from a cysteine residue to the thiazole ring in the thiamine intermediate. In this context, we carried out a cysteine quantification in THI1 and THI1(A140V) using electron spin resonance (ESR). These data showed that THI1(A140V) contains more sulfur-containing cysteines than THI1, indicating that the function as a sulfur donor is conserved, but the rate of donation reaction is somehow affected. Also, the bound compounds were isolated from both proteins and are present in different amounts in each protein. Unfolding studies presented differences in melting temperatures and also in the concentration of guanidine at which half of the protein unfolds, thus showing that THI1(A140V) has its conformational stability affected by the mutation. Hence, despite keeping its function in the early steps during the synthesis of TPP precursor, our studies have shown a decrease in the THI1(A140V) stability, which might be slowing down the biological activity of the mutant, and thus contributing to thiamin auxotrophy.