The water cavity of yeast thioredoxin 1 (yTrx1) is an ancestral, conserved structural element that is poorly understood. We recently demonstrated that the water cavity is involved in the complex protein dynamics that are responsible for catalytic-relevant event of coupling hydration, proton exchange, and motion at the interacting loops. Its main feature is the presence of the conserved polar residue, Asp24, which is buried in a hydrophobic cavity. Here, we evaluated the role of the solvation of Asp24 as the main element responsible for the formation of the water cavity in thioredoxins. We showed that the substitution of Asp24 to a hydrophobic residue (D24A) was not sufficient to completely close the cavity. The dynamics of the mutant D24A of yTrx1 at multiple timescales revealed that the mutant D24A presents motions at different timescales near the active site, interaction loops and water cavity, revealing the existence of a smaller dissected cavity. Molecular dynamics simulation, along with experimental molecular dynamics, enabled a detailed description of the water cavity in the wild type yTrx1 and D24A. The cavity connects the interacting loops, the central β-sheet, and α-helices 2 and 4. It is formed by three contiguous lobes, which we call lobes A, B and C. Lobe A is hydrophilic and the most superficial. It is formed primarily by the conserved Lys54. Lobe B is the central lobe formed by the catalytically important residues Cys33 and Asp24, which are strategically positioned. Lobe C is the most hydrophobic and is formed by the conserved cis-Pro73. The central lobe B is closed upon the mutation D24A, revealing that independent forces other than solvation of Asp24 maintain lobe A and C in the open configuration. These data allow us to better understand the properties of this enzyme.