P-glycoprotein (P-gp) plays a significant role in multi-drug resistance (MDR) of cancer cells, resulting in the failure of cancer chemotherapy. Lathyrane diterpene was a class of promising MDR modulator. The phytochemical investigation on the seeds of Euphorbia lathyris L. aff ;orded four new lathyrol-type diterpenoids (1-4), together with seventeen known ones (5-23). The structures of these compounds were elucidated by using 1D and 2D NMR spectroscopy. All the compounds were evaluated reversing MDR activity in HepG2/ADR cells. Compounds 2-4, 7-9, 11, 13-14, 16, 18-19, and 2 1-2 3 at 20 μM was able to reverse MDR of HepG2/ADR cells to adriamycin (reversal fold: 10.05–448.39). In the investigation of reversing the MDR mechanism, the most potent compound 21 facilitated the accumulation of intracellular adriamycin in HepG2/ADR cells in the time-dependent model. Although 21 neither affected the P-gp distribution nor expression in HepG2/ADR cells, the amount of P-gp monomer was induced by 21 and verapamil. Compound 21 has also activated the P-gp coupled ATPase activity in a dose-dependent model. The molecular docking analysis implied that 21 had lower binding energy than verapamil and adriamycin. The present data demonstrated that lathyrane diterpenes may act as a class of high-affinity P-gp substrate and was effluxed by P-gp monomer to reverse the MDR.