Skp1 is a nucleocytoplasmic protein that is post-translationally modified by a pentasaccharide, Ga1 alpha 1, Ga1 alpha 1, 3Fuc alpha 1, 2Gal beta 1, 3GlcNAc alpha 1O-, at a 4-hydroxylated derivative of Pro-143 in the amebazoan Dictyostelium discoideum. An enzymatic activity that catalyzes formation of the Ga1 alpha 1, 3Fuc linkage by transfer of Gal from UDP-alpha Gal to Fuc alpha 1, 2Gal beta 1, 3G1cNAc alpha 1O-benzyl, or the corresponding glycoform of Skp1, was described previously in cytosolic extracts of Dictyostelium. A protein GT78 associated with this activity has been purified to chromatographic homogeneity. In-gel tryptic digestion followed by nano-liquid chromatography-mass spectrometry on a quadrupole time-of-flight geometry instrument with data-dependent tandem mass spectrometry acquisition yielded a number of peptide fragmentation spectra, nine of which were manually de novo sequenced and found to map onto a predicted 3-exon gene of unknown function on chromosome 4. GT78 is predicted to comprise 648 amino acids with an N-terminal glycosyltransferase and a C-terminal beta-propeller domain. Overexpression of GT78 with a His(6)-tag resulted in a 120-fold increase in Ga1T-activity in cytosolic extracts, and purified His(6)-GT78 exhibited alpha 3Ga1T-activity toward a synthetic acceptor substrate. Expression of the truncated N-terminal region confirmed the predicted catalytic activity of this domain. Disruption of the GT78 gene led to a loss of enzyme activity in extracts and accumulation of the non-galactosylated isoform of Skp1 in cells. GT78 therefore represents the Skp1 alpha 3Ga1T, and its mechanism conforms to the sequential model of Skp1 glycosylation in the cytoplasm shown for earlier enzymes in the pathway. Informatics studies suggest that related catalytic domains are expressed in the Golgi or cytoplasm of plants, other protozoans, and animals.