Southwestern China, a biodiversity hotspot, underwent dramatic climate change following the Last Glacial Maximum and intensive human activity also exerted a major impact since the late Holocene. In this paper, we report a pollen record from Yilong Lake in Yunnan Province, southwestern China, to understand vegetation response to long-term climatic variation and human activities at a regional-level over the last 27,000 years. Findings show that temperate and coniferous taxa were dominant before 18 cal kyr BP reflecting the glacial climate. Between 18 and 14 cal kyr BP, thermophilic taxa increased in response to global deglaciation. A notable shift from deciduous to evergreen forests occurred at approximately 14 cal kyr BP, after which pollen concentration increased but pollen diversity decreased. Pollen assemblages indicated dense forests, primarily composed of evergreen oaks developed under warm and strong summer monsoon conditions from 13 to 8 cal kyr BP. High rates of vegetation change occurred during the middle Holocene before vegetation diversity gradually increased during the Holocene Thermal Optimum, with high variability of precipitation in the Yilong Lake region. Anthropogenic forcing progressively became the main factor affecting vegetation composition and vegetation change rates over the past three millennia.