Bupleurum falcatum L. has been used traditionally as a medicinal herb in Korean medicine. The hexane fraction of BF (HFBF), which was profiled with Direct Analysis in Real Time-Mass Spectrometry (DART-MS), activates the secretion of glucagon-like peptide-1 (GLP-1) in NCI-H716 cells significantly. We performed a microarray analysis and GLP-1 ELISA assay, as well as calcium imaging experiments with inhibitors, to investigate the mechanism of action of the HFBF. Through the microarray analysis, it was found that the ITPR2 gene that encodes the inositol 1,4,5-trisphosphate (IP3) receptor is up-regulated and the HFBF induces cell depolarization by inhibiting the voltage-gated channel expression in NCI-H716 cells. In addition, we found that the intracellular calcium in NCI-H716 cells, with Gallein, U73122, and 2APB as inhibitors, was decreased. These results suggest that the HFBF activates the GLP-1 secretion through the G βγ pathways in the enteroendocrine L cells after treatment with the HFBF.