Hyperglycemia can result from a relative or absolute lack of functional insulin secreted by the pancreatic β-cells. Prohormone processing enzymes play an essential role in the secretion of mature and fully functional insulin. Defects in insulin processing enzymes including prohormone convertases 1/3 and 2, and carboxypeptidase E (CPE) can lead to β-cell stress and hyperproinsulinemia, both of which are features of type 2 diabetes. Despite their importance, the regulation and role of this family of enzymes remain to be fully elucidated. Previously, we demonstrated that lipotoxicity led to the degradation of CPE, but did not affect its related enzyme, carboxypeptidase D (CPD). In this study, we found that CPD was significantly up-regulated by elevated glucose, while CPE was not. Low doses of insulin also increased CPD protein levels, consistent with a role for autocrine signaling. Glucose and insulin did not affect CPD or CPE expression in an α-cell line. Furthermore, insulin treatment altered the CPD sub-cellular localization, which was distinct from CPE. Somewhat surprisingly, the loss of CPE did not affect the levels of CPD. Knockdown of CPD exerted no effect on CPE protein levels. In addition, while our previous study demonstrated that even modest reduction of CPE was sufficient to induce β-cell apoptosis, CPD knockdown did not affect cell viability. Taken together, our data demonstrate that CPE and CPD are differentially localized, differentially regulated and unlikely to have compensatory functions in pancreatic β-cells.