Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) analogues are approved for treating type 2 diabetes, but are known to activate GLP-1R signaling globally and constitutively. Active compound N55, previously isolated from fenugreek, enhances the potency of GLP-1 without activating GLP-1R. Here we investigated if N55 lowers plasma glucose base on physiological levels of GLP-1. N55 was found to dose-dependently lower plasma glucose in non-fasted mice but not in the fasted mice, with the effect attenuated by GLP-1R antagonist exendin-(9–39) (Ex-9). On the other hand, when co-administered with dipeptidyl peptidase-IV (DPP4) -resistant [Aib8]-GLP-1(7–36) amide (GLP-1′), hypoglycemic response to N55 was observed in the fasted mice. This enhancement was also found to display dose dependency. N55 enhancement of the hypoglycemic and insulinotropic action of GLP-1′ was eliminated upon Ex-9 treatment. Both exendin-4 (Ex-4) and DPP4-resistant GLP-1 mutant peptide ([Aib8, E22, E30]-GLP-1(7–36) amide) activated GLP-1R and improved glucose tolerance but the enhancement effect of N55 was not observed in vivo or in vitro. In conclusions, N55 lowers plasma glucose according to prandial status by enhancing the response of physiological levels of GLP-1 and is much less likely to disrupt tight regulation of GLP-1R signaling as compare to GLP-1 analogues.