A thymidylate kinase catalyzes the reversible phosphoryl transfer from adenosine triphosphate (ATP) to thymidine monophosphate (dTMP) to form thymidine diphosphate (dTDP). This enzyme from Vibrio cholerae O1 biovar eltor str. N16961 was co-crystallized with dTMP and ADP. Crystal diffracted to 1.8 Å resolution and a molecular replacement solution was obtained using the structure of the Escherichia coli thymidylate kinase with the bisubstrate inhibitor TP5A (PDB ID 4TMK) as a model. Analysis of the two ligand-binding sites in the initial model of the V. cholerae protein revealed a positive peak close to the dTMP’s phosphate group. This density clearly indicated presence of an additional phosphate group, which however seemed to be linked to the dTMP’s phosphate group. Knowing the protein’s function and assuming cellular uptake of dTDP can occur during purification procedure, this nucleotide was modeled with half occupancy into the dTMP-binding site. No negative difference density was observed after refinement. That is, the crystal structure of the V. cholerae thymidylate kinase has been solved in complex with dTMP, dTMDP and ADP. Two calcium ions interact with the nucleotides. Four more calcium ions are found associated with the protein as a result of presence of 0.2 M CaCl2 in the crystallization condition.