Two new neolignans and one new lignan (1–3) were obtained from the roots of Paeonia lactiflora . Their structures were unambiguously elucidated based on extensive spectroscopic analysis, single-crystal X-ray crystallography, and the calculated and experimental electronic circular dichroism (ECD) spectra. Compound 1 was a racemic mixture and successfully resolved into the anticipated enantiomers via chiral-phase HPLC. Compound 3 demonstrated moderate inhibitory activity against human carboxylesterase 2A1 (hCES2A1) with an IC 50 value of 7.28 ± 0.94 μmol· −1 .