Vegetation and fire activity have dynamically changed in response to past variations in global and regional climate. Here we investigate these responses across the Neotropics based on the analysis of modern vegetation distribution and fire activity in relation to modern climate patterns in the one hand, and a compilation of 243 vegetation records and 127 charcoal records encompassing the last 21,000 years before present (ka) in relation to past climate changes on the other hand. Our analyses on the dynamics of past tree cover and fire activity focus on seven subregions: (1) northern Neotropics (NNeo); (2) central Andes (CAn); (3) Amazonia; (4) northeastern Brazil (NEB); (5) central-eastern Brazil (CEB); (6) southeastern South America (SESA); and (7) southern Andes (SAn). The regionalized assessment unveils spatial heterogeneity in the timing and controls of vegetation and fire dynamics. Temperature, atmospheric CO₂ concentrations, and precipitation exhibit distinct and alternating roles as primary drivers of tree cover and fire regime changes. During the Last Glacial Maximum (LGM, here covering 21–19 ka), biomass growth in high elevation sites (CAn) and in sub- and extra-tropical latitudes (SESA and SAn) was mainly limited by low temperatures and atmospheric CO₂ concentrations, while fuel-limited conditions restrained fire activity. In warmer tropical regions (NNeo, Amazonia, CEB), moisture availability was likely the main controlling factor of both vegetation and fire. Throughout the deglacial phase (19–11.7 ka), progressive warming fostered a gradual biomass expansion, leading to an intensification of fire activity in the sub- and extra-tropical temperature-limited regions. Meanwhile, increased (decreased) precipitation associated with millennial-scale events favored increases (decreases) in tree cover in CAn, Amazonia, CEB, and NEB (NNeo). Between 14–13 ka, most southern latitude subregions (Amazonia, CEB, SESA, SAn) saw a stepwise rise in fire activity coeval with a second rapid warming, contrary to decreased fire activity in NNeo amid relatively wetter conditions. Throughout the Holocene, when temperature and atmospheric CO₂ fluctuations were lower, shifts in precipitation became the primary driver of vegetation and fire dynamics across all the Neotropics. The intensification of the South American Summer Monsoon throughout the Holocene favored a continuous increase in tree cover over Amazonia, CEB, and SESA, but led to a forest cover decrease in NNeo and NEB. From the early- to the mid-Holocene, the strengthening of the Southern Westerly Winds promoted vegetation expansion and fire regime weakening in SAn. In the late Holocene, human impacts became more pronounced, with a clearer effect on regional tree cover and fire activity, particularly in NNeo and CAn.