Fire is a powerful tool for conservation management at a landscape scale, but a rigorous evidence base is often lacking for understanding its impacts on biodiversity in different biomes. Fire-induced changes to habitat openness have been identified as an underlying driver of responses of faunal communities, including for ants. However, most studies of the impacts of fire on ant communities consider only epigeic (foraging on the soil surface) species, which may not reflect the responses of species inhabiting other vertical strata. Here, we examine how the responses of ant communities vary among vertical strata in a highly fire-prone biome. We use a long-term field experiment to quantify the effects of fire on the abundance, richness, and composition of ant assemblages of four vertical strata (subterranean, leaf litter, epigeic, and arboreal) in an Australian tropical savanna. We first document the extent to which each stratum harbors distinct assemblages. We then assess how the assemblage of each stratum responds to three fire-related predictors: fire frequency, fire activity, and vegetation cover. Each stratum harbored a distinct ant assemblage and showed different responses to fire. Leaf litter and epigeic ants were most sensitive to fire because it directly affects their microhabitats, but they showed contrasting negative and positive responses, respectively. Subterranean ants were the least sensitive because of the insulating effects of soil. Our results show that co-occurring species of the same taxonomic group differ in the strength and direction of their response to fire depending on the stratum they inhabit. As such, effective fire management for biodiversity conservation should consider species in all vertical strata.