Carboxypeptidase O (CPO) is a member of the M14 family of metallocarboxypeptidases with a preference for the cleavage of C-terminal acidic amino acids. CPO is largely expressed in the small intestine, although it has been detected in other tissues such as the brain and ovaries. CPO does not contain a prodomain, nor is it strongly regulated by pH, and hence appears to exist as a constitutively active enzyme. The goal of this study was to investigate the intracellular distribution and activity of CPO in order to predict physiological substrates and function. The distribution of CPO, when expressed in MDCK cells, was analyzed by immunofluorescence microscopy. Soon after addition of nutrient-rich media, CPO was found to associate with lipid droplets, causing an increase in lipid droplet quantity. As media became depleted, CPO moved to a broader ER distribution, no longer impacting lipid droplet numbers. Membrane cholesterol levels played a role in the distribution and in vitro enzymatic activity of CPO, with cholesterol enrichment leading to decreased lipid droplet association and enzymatic activity. The ability of CPO to cleave C-terminal amino acids within the early secretory pathway (in vivo) was examined using Gaussia luciferase as a substrate, C-terminally tagged with variants of an ER retention signal. While no effect of cholesterol was observed, these data show that CPO does function as an active enzyme within the ER where it removes C-terminal glutamates and aspartates, as well as a number of polar amino acids.