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Forest-grassland mosaics, with abrupt boundaries between the two vegetation types, occur across the globe. Fire and herbivory are widely considered primary drivers that maintain these mosaics by limiting tree establishment in grasslands, while edaphic factors and frosts are generally considered to be secondary factors that reinforce these effects. However, the relative importance of these drivers likely varies across systems. In particular, although frost is known to occur in many montane tropical mosaics, experimental evidence for its role as a driving factor is limited. We used replicated in situ transplant and warming experiments to examine the role of microclimate (frost and freezing temperatures) and soil in influencing germination and seedling survival of both native forest trees and alien invasive Acacia trees in grasslands of a tropical montane forest-grassland mosaic in the Western Ghats of southern India. Seed germination of both native and alien tree species was higher in grasslands regardless of soil type, indicating that germination was not the limiting stage to tree establishment. However, irrespective of soil type, native seedlings in grasslands incurred high mortality following winter frosts and freezing temperatures relative to native seedlings in adjoining forests where freezing temperatures did not occur. Seedling survival through the tropical winter was thus a primary limitation to native tree establishment in grasslands. In contrast, alien Acacia seedlings in grasslands incurred much lower levels of winter mortality. Experimental night-time warming in grasslands significantly enhanced over-winter survival of all tree seedlings, but increases were much greater for alien Acacia than for native tree seedlings. Synthesis. Our results provide evidence for a primary role for frost and freezing temperatures in limiting tree establishment in grasslands of this tropical montane forest-grassland mosaic. Future increases in temperature are likely to release trees from this limitation and favour tree expansion into grasslands, with rates of expansion of non-native Acacia likely to be much greater than that of native trees. We suggest that studies of frost limitation to plant establishment are needed across a range of tropical ecosystems to re-evaluate the general importance of frost as a driver of vegetation transitions in the tropics.