It has been widely recognized that mammal brain size predominantly increases over evolutionary time. Safi et al. [Biol Lett 2005;1:283-286] questioned the generality of this trend, arguing that brain size evolution among bats involved reduction in multiple lineages as well as enlargement in others. Our study explored the direction of change in the evolution of bat brain size by estimating brain volume in fossil bats, using synchrotron radiation X-ray tomographic microscopy. Virtual endocasts were generated from 2 Hipposideros species: 3 specimens of Oligocene Hipposideros schlosseri (∼35 Ma) and 3 of Miocene Hipposideros bouziguensis (∼20 Ma). Upper molar tooth dimensions (M(2) length × width) collected for 43 extant insectivorous bat species were used to derive empirical formulae to estimate body mass in the fossil bats. Brain size was found to be relatively smaller in the fossil bats than in the average extant bat both with raw data and after allowing for phylogenetic inertia. Phylogenetic modeling of ancestral relative brain size with and without fossil bats confirmed a general trend towards evolutionary increase in this bat lineage.