The metalloprotease meprin β cleaves the amyloid precursor protein (APP) as a β-secretase, thereby releasing neurotoxic Aβ peptides, a hallmark in sporadic Alzheimer’s disease (AD). We found a significant increase in meprin β protein levels in AD brain tissue. Additionally, we demonstrate the in vivo relevance of meprin β with increased endogenous sAPPα levels in the brains of meprin β knock-out mice, as a consequence of increased substrate availability for the competitive α-secretase. Therefore, regulation of meprin β activity might be a suitable strategy for the treatment of AD. Meprin β is a type 1 transmembrane protein, but can be shed from the cell surface by ADAM10/17. Importantly, only the membrane tethered protease acts as a β-secretase. Pancreatic trypsin and human tissue kallikrein-related peptidases 4, 5 and 8 were found to activate soluble meprin β. However, regarding the structural properties of full-length meprin β, we hypothesized that the buried activation site in the propeptide, which is in close proximity to the plasma membrane, is shielded towards soluble tryptic proteases and rather requires membrane bound activators. Indeed, we identified the transmembrane serine protease matriptase-2 (MT2) as a new potent activator of membrane bound meprin β, analyzed by specific fluorogenic peptide cleavage assay, biotinylation experiments, and confocal laser scanning microscopy. We show that specifically MT2, but not another membrane bound serine protease or pancreatic trypsin, is capable of activating full-length meprin β at the cell surface, required for its shedding activity and subsequent APP cleavage at the β-secretase site.