Land use changes (LUC) from pasture to sugarcane (Saccharum spp.) crop are expected to add 6.4 Mha of new sugarcane land by 2021 in the Brazilian Cerrado and Atlantic Forest biomes. We assessed the effects of these LUC on the abundance and community structure of animals that inhabit soils belowground through a field survey using chronosequences of land uses comprising native vegetation, pasture, and sugarcane along a 1000-km-long transect across these two major tropical biomes in Brazil. Macrofauna community composition differed among land uses. While most groups were associated with samples taken in native vegetation, high abundance of termites and earthworms appeared associated with pasture soils. Linear mixed effects analysis showed that LUC affected total abundance (X2(1) = 6.79, p = 0.03) and taxa richness (X2(1) = 6.08, p = 0.04) of soil macrofauna. Abundance increased from 411 ± 70 individuals m− 2 in native vegetation to 1111 ± 202 individuals m− 2 in pasture, but decreased sharply to 106 ± 24 individuals m− 2 in sugarcane soils. Diversity decreased 24% from native vegetation to pasture, and 39% from pasture to sugarcane. Thus, a reduction of ~ 90% in soil macrofauna abundance, besides a loss of ~ 40% in the diversity of macrofauna groups, can be expected when sugarcane crops replace pasture in Brazilian tropical soils. In general, higher abundances of major macrofauna groups (ants, coleopterans, earthworms, and termites) were associated with higher acidity and low contents of macronutrients and organic matter in soil. This study draws attention for a significant biodiversity loss belowground due to tropical LUC in sugarcane expansion areas. Given that many groups of soil macrofauna are recognized as key mediators of ecosystem processes such as soil aggregation, nutrients cycling and soil carbon storage, our results warrant further efforts to understand the impacts of altering belowground biodiversity and composition on soil functioning and agriculture performance across LUC in the tropics.