Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess. The medium for its axenic culture contains glucose as energy source, and we addressed the question whether E. histolytica can also use fructose instead. As the amoebic hexokinases do not phosphorylate fructose, a separate fructokinase is essential. The genome project revealed a single candidate gene encoding an E. histolytica homolog of bacterial fructokinases. This gene was cloned, and the recombinant enzyme had a magnesium-dependent fructose 6-kinase activity (EC 2.7.1.4) with a K m for fructose of 0.156 mM and a V max of 131 U/mg protein. Recombinant fructokinase also showed a much weaker mannokinase activity, but no activity with glucose or galactose. The amoebae could be switched from glucose to fructose medium without any detectable consequence on doubling time. Fructokinase messenger RNA (mRNA) was modestly but significantly upregulated in amoebae switched to fructose medium as well as in fructose-adapted E. histolytica.