CD147 is a widely expressed matrix metalloproteinase inducer involved in the regulation of cell migration. The high glycosylation and ability to undergo oligomerization have been linked to CD147 function, yet there is limited understanding on the molecular mechanisms behind these processes. The current study demonstrates that the expression of Golgi α1,2-mannosidase I is key to maintaining the cell surface organization of CD147 during cell migration. Using an in vitro model of stratified human corneal epithelial wound healing, we show that CD147 is clustered within lateral plasma membranes at the leading edge of adjacent migrating cells. This localization correlates with a surge in matrix metalloproteinase activity and an increase in the expression of α1,2-mannosidase subtype IC (MAN1C1). Global inhibition of α1,2-mannosidase I activity with deoxymannojirimycin markedly attenuates the glycosylation of CD147 and disrupts its surface distribution at the leading edge, concomitantly reducing the expression of matrix metalloproteinase-9. Likewise, treatment with deoxymannojirimycin or siRNA-mediated knockdown of MAN1C1 impairs the ability of the carbohydrate-binding protein galectin-3 to stimulate CD147 clustering in unwounded cells. We conclude that the mannose-trimming activity of α1,2-mannosidase I coordinates the clustering and compartmentalization of CD147 that follows an epithelial injury.