One achiral tetra-aryl cyclobutane [rheundulin A (1)] and three stilbene glycosides [rheundulins B-D (2-4)] were isolated from the methanol extract of Rheum undulatum L., along with eight known compounds (5-12). Structural determination of the new compounds (1-4) was accomplished using comprehensive spectroscopic methods. Compound 1 represents the first example of a dimeric stilbene linked via a cyclobutane ring from the Rheum genus. All isolates were screened for their inhibition against α-glucosidase. Among them, stilbene derivatives (5 and 6) showed strong inhibitory effects on α-glucosidase with IC<sub>50</sub> values of 0.5 and 15.4 µM, respectively, which were significantly higher than that of the positive control, acarbose (IC<sub>50</sub> = 126.8 µM). Rheundulin A (1) showed moderate α-glucosidase inhibition with an IC<sub>50</sub> value of 80.1 µM. In addition, kinetic analysis and molecular docking simulation of the most active compound (5) with α-glucosidase were performed for the first time. Kinetic studies revealed that compound 5 competitively inhibited the active site of α-glucosidase (K<sub>i</sub> = 0.40 µM), while 6 had a mixed-type inhibitory effect against α-glucosidase (K<sub>i</sub> = 15.34 µM). Molecular docking simulations of 5 and 6 demonstrated negative-binding energies, indicating high proximity to the active site and tight binding to α-glucosidase enzyme.