Metastasis (spread of cancer cells) is the major cause of cancer related deaths. The reason why cancer cells spreads is largely unknown, but proteinases have been suggested to take part in this process. The proteinases have also emerged as promising to utilize in cancer therapy. However, the lack of pre-established knowledge has previously led to failure in clinical trials, e.g. inhibitors against matrix metalloproteinases (MMPs) caused severe side effects in patients. The cysteine proteinase legumain is overexpressed in many solid human tumors, and overexpression is associated with enhanced metastasis. Currently, the knowledge about subcellular localization, trafficking and requirements for legumain activation remains largely unexplored. Known factors that influence the transport and activation of proteins in general are glycosylation and phosphorylation. Both of the aforementioned modifications have been demonstrated on legumain, but the functional role remains unknown. In this study, we investigated processing and localization of legumain after manipulation of glycosylation and phosphorylation. The colorectal cell lines HCT116 and SW620 were used as cell models. N-linked glycosylation was inhibited by the biochemical tool tunicamycin, whereas phosphorylation was blocked by staurosporine or H7. Legumain expression, processing and distribution were analyzed by immunoblotting and confocal microscopy. The results have shown that processing of legumain to the mature active form was totally absent after tunicamycin treatment. It was also identified that tunicamycin resulted in a decreased nuclear transport, whereas legumain secretion was apparently not affected. Furthermore, treatment with staurosporine changed the cell morphology, but this was not observed after optimizing the experiment with H7. However, neither staurosporine nor H7 seemed to exert influences on legumain expression or processing. Overall the results imply that glycosylation, but not phosphorylation, is essential for legumain processing. Furthermore, glycosylation affects legumain transport to the nucleus, but not to the extracellular environment. However, additional research needs to be done to conclude how glycosylation affects intracellular legumain transport.