Administration of an alpha-glucosidase inhibitor, voglibose, increases the secretion of glucagon-like peptide (GLP)-1, a key modulator of pancreatic islet hormone secretion and glucose homeostasis. In the present study, novel mechanisms by which voglibose increases active GLP-1 circulation were evaluated. Voglibose (0.001 and 0.005%) was administered in the diet to ob/ob mice for 1 day or 3 to 4 weeks to determine effects on incretin profiles and plasma activity of dipeptidyl peptidase-4 (DPP-4), an enzyme responsible for GLP-1 degradation. Voglibose showed no direct inhibitory effect against DPP-4 in vitro (DPP-4 inhibitor alogliptin, IC(50) < 10 nM). Likewise, 1-day treatment with voglibose did not change plasma DPP-4 activity; however, it increased plasma active GLP-1 by 1.6- to 3.4-fold. After chronic treatment, voglibose stimulated GLP-1 secretion, as evidenced by the 1.3- to 1.5-fold increase in plasma active plus inactive amidated GLP-1 levels. Plasma DPP-4 activity was decreased unexpectedly by 40 to 51%, resulting from reduced plasma DPP-4 concentrations in voglibose-treated mice. Voglibose increased GLP-1 content by 1.5- to 1.6-fold and 1.4- to 1.6-fold in the lower intestine and colon, respectively. The increased GLP-1 content in the colon was associated with elevated expression of gut glucagon gene. Chronic treatment with voglibose resulted in 1.9- to 4.1-fold increase in active GLP-1 circulation, which was higher than 1-day treatment. A similar treatment with pioglitazone (0.03%), an insulin sensitizer, did not affect plasma DPP-4 activity or GLP-1 levels. These results suggest that increased GLP-1 secretion, decreased DPP-4 activity, and increased gut GLP-1 content may have contributed to increased active GLP-1 circulation after chronic treatment with voglibose in a glucose control-independent manner in ob/ob mice.