Escherichia coli isocitrate dehydrogenase (IDH) is regulated by reversible phosphorylation on Ser113. Latest phosphoproteomic studies revealed that eukaryotic IDHs can also be phosphorylated on the analogous Ser site. So as to understand the possible phosphorylation mechanism, the equivalent Ser of NADP-IDHs from yeast Saccharomyces cerevisiae (ScIDH) and Yarrowia lipolytica(YlIDH) were investigated by site-directed mutagenesis. ScIDH Ser110 and YlIDH Ser103 were replaced by Asp or Glu to mimic a continuous phosphorylation state. Meanwhile, the effects of another four amino acids (Thr, Tyr, Gly, Ala) with various side chain on IDH activity were determined as well. Enzymatic analysis showed that replacement of Ser with Asp or Glu nearly inactivated ScIDH and YlIDH. Four other mutant enzymes of ScIDH, S110T, S110G, S110A, and S110Y, retained 38.07%, 3.24%, 2.65%, and 0.01% of its original activity, and four other mutant enzymes of YlIDH, S103T, S103G, S103A, and S103Y retained 44.26%, 27.99%, 16.29%, and 0.01% of its original activity, respectively. These results suggested that phosphorylation on eukaryotic IDHs has identical consequence to that on the bacterial IDHs. We thus presume that phosphorylation on the substrate-binding Ser shall be a common regulatory mechanism among IDHs.