Aim The forest steppe of the Transylvanian Plain is a landscape of exceptionally diverse steppe-like and semi-natural grasslands. Is this vegetation a remnant of a once continuous temperate forest extensively cleared by humans, or has the area, since the last glacial, always been a forest steppe? Understanding the processes that drive temperate grassland formation is important because effective management of this biome is critical to the conservation of the European cultural landscape. Location Lake Stiucii, north-western Romania, central-eastern Europe. Methods We analysed multi-proxy variables (pollen, coprophilous fungi, plant macroremains, macrocharcoal) from a 55,000 year discontinuous sequence (c. 55,000–35,000; 13,000–0 cal. yr bp), integrating models of pollen-based vegetation cover, biome reconstruction, global atmospheric simulations and archaeological records. Results Needleleaf woodland occurred during glacial Marine Isotope Stage (MIS) 3, but contracted at the end of this period. Forest coverage of c. 55% (early Holocene) and 65% (mid-Holocene) prevailed through the Holocene, but Bronze Age humans extensively cleared forests after 3700 cal. yr bp. Forest coverage was most widespread between 8600 and 3700 cal. yr bp, whereas grasses, steppe and xerothermic forbs were most extensive between 11,700 and 8600 cal. yr bp and during the last 3700 cal. yr bp. Cerealia pollen indicate the presence of arable agriculture by c. 7000 cal. yr bp. Main conclusions We have provided the first unequivocal evidence for needleleaf woodland during glacial MIS 3 in this region. Extensive forests prevailed prior to 3700 cal. yr bp, challenging the hypothesis that the Transylvanian lowlands were never wooded following the last glaciation. However, these forests were never fully closed either, reflecting dry growing season conditions, recurrent fires and anthropogenic impacts, which have favoured grassland persistence throughout the Holocene. The longevity of natural and semi-natural grasslands in the region may explain their current exceptional biodiversity. This longer-term perspective implies that future climatic warming and associated fire will maintain these grasslands.