There has been an ongoing debate about human impacts on the evolution of the vegetation cover on the Yangtze delta plain dating back to the Neolithic period. In this study, we carried out pollen identification and grain size analysis on two sediment profiles obtained from the Neolithic Guangfulin site in the Yangtze delta. Together with published results of radiocarbon ages, organic elemental chemistry and archaeological findings, we reconstructed the palaeoecological evolution and human activities at the site during the mid- to late Holocene and distinguished between the impacts on vegetation induced by human and hydrological processes. The results show three events of significant reduction in the abundance of arboreal pollen, which occurred at c. 4635, 2000, and 800 cal yr BP, respectively. The first two events were accompanied by an obvious increase in Poaceae pollen, whereas the last one occurred with a sharp increase in the abundance of Brassicaceae pollen. These findings suggest that the reductions in arboreal pollen resulted from deforestation for the expansion of rice cultivation at c. 4635 and 2000 cal yr BP, and expansion of Brassicaceous oil crops c. 800 years ago. The change in cultivation pattern c. 800 years ago was consistent with the increase in population migration from northern China caused by war at that time. The pollen of aquatic plants increased sharply at c. 4500 cal yr BP, which reflected the change in hydrological environment related to sea-level rise at the Yangtze River mouth.