The human mannose 6-phosphate uncovering enzyme participates in the uncovering of the mannose 6-phosphate recognition tag on lysosomal enzymes, a process that facilitates recognition of those enzymes by mannose 6-phosphate receptors to ensure delivery to lysosomes. Uncovering enzyme has been identified on the trans-Golgi network at steady state. It has been shown to traffic to the plasma membrane from where it is rapidly internalized via endosomal structures, the process being mediated by a tyrosine-based internalization motif, Y488HPL, in its cytoplasmic tail. Using immunogold electron microscopy a GFP-uncovering enzyme fusion construct was found to be colocalized with the cation-dependent mannose 6-phosphate receptor in regions of the trans-Golgi network, suggesting that uncovering enzyme might follow a similar pathway of exit from the trans-Golgi network as that of the cation-dependent mannose 6-phosphate receptor. In this study, we identified the signal sequence in the cytoplasmic tail of uncovering enzyme responsible for its exit from the trans-Golgi network. Using GFP fusion constructs of the transmembrane and cytoplasmic domains of uncovering enzyme, we could show, by automated analysis of confocal immunofluorescence images, that residues Q492EMN in the cytoplasmic tail of uncovering enzyme are involved in its exit from the trans-Golgi network. Detailed characterization of the exit signal revealed that residue Q492 is the most important to the exit function while M494 and N495 also contribute. The cytoplasmic tail of the uncovering enzyme does not possess any of the known canonical signal sequences for interaction with Golgi-associated gamma ear-containing adaptor proteins. The identification of a trans-Golgi network exit signal in its cytoplasmic tail elucidates the trafficking pathway of uncovering enzyme, a crucial player in the process of lysosomal biogenesis.