In a continuing search for potential inhibitors against human carboxylesterases 1A1 and 2A1 (hCES1A1 and hCES2A1), an EtOAc extract of the roots of Paeonia lactiflora showed strong hCES inhibition activity. Bioassay-guided fractionation led to the isolation of 26 terpenoids including 12 new ones (1-5, 7-12, and 26). Among these, sesquiterpenoids 1 and 6, monoterpenoids 10, 11, and 13-15, and triterpenoids 18-20, 22, and 24-26 contributed to the hCES2A1 inhibition, in the IC range of 1.9-14.5 μM, while the pentacyclic triterpenoids 18-26 were responsible for the potent inhibitory activity against hCES1A1, with IC values less than 5.0 μM. The structures of all the compounds were elucidated using MS and 1D and 2D NMR data, and the absolute configurations of the new compounds were resolved via specific rotation, experimental and calculated ECD spectra, and single-crystal X-ray diffraction analysis. The structure-activity relationship analysis highlighted that the free HO-3 group in the pentacyclic triterpenoids is crucial for their potent inhibitory activity against hCES1A1.