Trypanosoma brucei, the etiological agent of sleeping sickness, has particular peroxisome-like organelles called glycosomes where several metabolic pathways are compartmentalized, including glycolysis. Many enzymes from sugar nucleotide biosynthetic pathways are located in this organelle, contrasting with the cytosolic localisation reported in the eukaryota domain. The UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for UDP-glucose (UDP-Glc) production, is essential for growth and survival of trypanosomes. In addition, UGP is localized in both glycosomes and cytosol, raising questions about why would these parasites maintain a pathway functional in two different subcellular compartments. UGP is imported into glycosomes by piggybacking on the glycosomal protein PEPCK, which is the first evidence of piggybacking involving two proteins not functionally related, and the first report in trypanosomatids. The dataset demonstrates that UGP is present in the total cellular extract and the glycocomal fraction of the wild-type trypanosomes, while only in the total cellular extract of the PEPCK null mutant.