The southwestern Amazon Rainforest Ecotone (ARE) is the transitional landscape between the tropical forest and seasonally flooded savannahs of the Bolivian Llanos de Moxos. These heterogeneous landscapes harbour high levels of biodiversity and some of the earliest records of human occupation and plant domestication in Amazonia. While persistent Indigenous legacies have been demonstrated elsewhere in the Amazon, it is unclear how past human–environment interactions may have shaped vegetation composition and structure in the ARE. Here, we examine 6000 years of archaeological and palaeoecological data from Laguna Versalles (LV), Bolivia. LV was dominated by stable rainforest vegetation throughout the Holocene. Maize cultivation and cultural burning are present afterca5700 cal yr BP. Polyculture cultivation of maize, manioc and leren afterca3400 cal yr BP predates the formation of Amazonian Dark/Brown Earth (ADE/ABE) soils (approx. 2400 cal yr BP). ADE/ABE formation is associated with agroforestry indicated by increased edible palms, includingMauritia flexuosaandAttaleasp., and record levels of burning, suggesting that fire played an important role in agroforestry practices. The frequent use of fire altered ADE/ABD forest composition and structure by controlling ignitions, decreasing fuel loads and increasing the abundance of plants preferred by humans. Cultural burning and polyculture agroforestry provided a stable subsistence strategy that persisted despite pronounced climate change and cultural transformations and has an enduring legacy in ADE/ABE forests in the ARE.This article is part of the theme issue ‘Tropical forests in the deep human past’.