Background: Visfatin represents an adipokine of the visceral adipose tissue exerting pleiotropic effects. The aim of the study was to characterize the physiological regulation of visfatin release in vivo in healthy, nondiabetic probands and in adipocytes in vitro. Subjects and Methods: One hundred healthy subjects (64 females and 36 males) underwent an oral glucose tolerance test (75 g). Probands were characterized by anthropometric and laboratory parameters. Fasting and postprandial (1 and 2 h) serum concentrations of glucose, insulin, and visfatin were measured. Stimulation experiments using glucose, insulin, mannitol, and glucagon-like peptide-1 (GLP-1) were performed on 3T3-L1 adipocytes including Western blot analysis. Results: Fasting visfatin levels were not different between males/females or lean/obese individuals but were negatively (r = −0.5; P < 0.001) correlated with fasting glucose levels. Visfatin levels were rapidly and significantly suppressed (P < 0.001) upon an oral glucose intake, and this suppression was more pronounced in overweight and female probands. In vitro experiments demonstrated that hyperglycemia, osmotic stress, and sex steroids did not influence visfatin release. In contrast, insulin strongly (P = 0.002) inhibited visfatin release in vitro by approximately 50%, and this suppression was more pronounced under hyperglycemia. Importantly, GLP-1 strongly (P < 0.001) inhibited adipocytic visfatin release by approximately 50%. Conclusions: Insulin and GLP-1 are responsible for the rapid suppression of visfatin levels upon an oral glucose uptake in healthy probands. The inhibitory effect of GLP-1 on adipocytic visfatin release together with the absence of direct glucose effects on visfatin release suggests the existence of a novel incretin-like effect represented by a GLP-1/visfatin/axis.