In order to investigate the diterpenoid components from the leaves of Pieris japonica and their acetylcholinesterase (AChE) inhibitory activities, with the aid of thin-layer chromatography color characteristics, silica gel, MCI and semi-preparative high-performance liquid chromatography technology, the target constituents were isolated and purified. The structures of the obtained compounds were identified by analyses of their spectral data (NMR and MS) and comparison of their data with those of reported in the literature. Meanwhile, AChE inhibitory effects of obtained diterpenoids were evaluated for the first time by a Ellman method. The results showed that eight diterpenoid compounds were isolated and identified from the leaves of P. japonica, namely pieriformoside F (1), 3-epi-grayanotoxin ⅩⅧ (2), 3-epi-grayanotoxin B (3), asebotoxin-X (4), pieriformosin B (5), asebotoxin Ⅲ (6), rhodojaponin Ⅲ (7), and pieriformosin C (8). Among them, Compound 1 was isolated from this plant for the first time, and Compound 8 showed AChE inhibitory activity. In conclusion, the results enriches the diterpenoids and bioactive components of P. japonica, which provides a certain theoretical basis for its further development and utilization.