[Objective] MilA, a cytidine 5′-monophosphate (CMP) hydroxymethylase which can convert CMP to 5-hydroxymethylcytidine-5′-monophosphate (HmCMP), was discovered and characterized in the mildiomycin biosynthetic pathway. MilA belongs to the superfamily of thymidylate synthase (TS) and cytidylate hydroxymethylase (CH), and was the only enzyme to date that was able to efficiently convert CMP other than dCMP to HmCMP. Three-dimensional structure prediction of MilA revealed high similarity to that of CH and TS. Ser94 of CH was proved to be a key residue mediating hydroxylation of the 5′-methyl cytosine of dCMP, corresponding residue is Thr102 in MilA and Pro92 in TS, respectively. In order to study the role of Thr102 in MilA, we constructed mutants MilA T102V and MilA T102L. [Methods] Based on above analysis, Thr102 of MilA was site-mutated into valine (Val, V) and leucine (Leu, L) to assay their catalytic efficiency. [Results] Compared to the wild type, activity of MilA T102V for CMP was significantly reduced by 87.1%, while that for dCMP was maintained at a similar level. By contrast, MilA T102L could not use CMP and dCMP any more. As for MilA T102L, the side chain of Leu102 was one carbon atom longer than both of Val102 and Thr102, implying that steric hindrance effect was involved in the substrate binding by MilA T102L. [Conclusion] The results demonstrated that Thr102 in MilA was essential for HmCMP formation. The function of MilAT102 may have reached optimization after naturally evolutionary fine-tuning.