This study aimed to investigate the role of angiotensin-converting enzyme 2 (ACE2) in regulating glucose homeostasis. Immunohistochemistry was used to investigate ACE2 expression in the pancreas. Glucose tolerance test, insulin secretion test, and insulin tolerance test were performed in age-matched male ACE2 knockout (KO) and wild-type (WT) mice. We found that ACE2 was positively expressed in the pancreas. Male ACE2 KO mice displayed a selective decrease in first-phase insulin secretion in response to glucose and a progressive impairment of glucose tolerance compared with age- and sex-matched WT mice. On the other hand, insulin sensitivity of the peripheral tissue in age-matched ACE2 KO and WT mice showed no difference. These findings suggest that ACE2 might play an important role in glucose homeostasis as well as type 2 diabetes.