Symbioses involving animals and chemoautotrophic bacteria form the foundation of entire ecosystems at deep-sea hydrothermal vents and cold seeps, but have so far not been reported in terrestrial or freshwater environments. A rare example of a terrestrial ecosystem sustained by chemoautotrophy is found within the sulfide-rich Frasassi limestone cave complex of central Italy. In this study, we report the discovery of abundant filamentous bacteria on the exoskeleton of Niphargus ictus, a macroinvertebrate endemic to Frasassi. Using 16S rDNA sequencing and fluorescence in situ hybridization (FISH), we show that N. ictus throughout the large cave complex are colonized by a single phylotype of bacteria in the sulfur-oxidizing clade Thiothrix. The epibiont phylotype is distinct from Thiothrix phylotypes that form conspicuous biofilms in the cave streams and pools inhabited by N. ictus. Using a combination of 13C labeling, FISH, and secondary ion mass spectrometry (SIMS), we show that the epibiotic Thiothrix are autotrophic, establishing the first known example of a non-marine chemoautotroph-animal symbiosis. Conditions supporting chemoautotrophy, and the N. ictus-Thiothrix association, likely commenced in the Frasassi cave complex between 350,000 and 1 million years ago. Therefore, the N. ictus-Thiothrix symbiosis is probably significantly younger than marine chemoautotrophic symbioses, many of which have been evolving for tens to hundreds of million years.