This study conducted in vivo and in situ experiments with rats to investigate the glucagon-like peptide-1 (GLP-1) secretion in response to oral or ileal administration of α-glucosyl-isoquercitrin (20–40 mmol in 2 mL; Q3G), fructooligosaccharides (200 mmol in 2 mL; FOS) and Q3G + FOS. Direct effects on GLP-1-producing L-cells were also examined by an in vitro study using a murine enteroendocrine cell line, GLUTag. To evaluate the plasma GLP-1 level, blood samples from jugular cannula for in vivo and portal cannula for in situ experiments were obtained before and after administration of Q3G, FOS, or Q3G + FOS. We found tendencies for increases but transient stimulation of GLP-1 secretion by Q3G in in vivo and in situ experiments. Although FOS alone did not have any effects, Q3G + FOS enhanced and prolonged high plasma GLP-1 level in both experiments. In addition, application of Q3G on GLUTag cells stimulated GLP-1 secretion while FOS enhanced the effect of Q3G. Our results suggest that Q3G + FOS possess the potential for the management or prevention of Type 2 diabetes mellitus (T2DM) by enhancing and prolonging the GLP-1 secretion via direct stimulation of GLP-1 producing L-cell.