ABSTRACTGrotta Polesini is one of the most famous paleontological and archaeological sites of central Italy, which testifies to its human occupation during the Lateglacial. The site comprises a cave system where systematic excavation campaigns have been carried out since the 1950s. In 1974, 656 mammal remains were collected but never studied. This fossil collection is here described for the first time through taxonomic and stable isotope analyses of the enamel of selected mammal teeth. The aim is to reconstruct the paleoenvironmental and climatic conditions of the site and to offer new information on terrestrial ecosystems during the Lateglacial in central Italy. The faunal assemblage studied herein, in addition to other species reported in previous works, suggests cold climate conditions. We also describe a right radius of an adult individual of Homo sapiens, increasing the human fossil record of the site. Carbon isotope data point to a scenario dominated by C3 plants in open and dry habitats, such as grasslands and steppes, in accordance with the pollen data from central Italy. The oxygen isotope data suggest the use of water resources with a local origin, i.e. local precipitation and surface waters with a provenance from the nearby Apennine chain. The ecology of the taxa influenced the oxygen isotope values, especially in the case of semi‐obligate to non‐obligate drinker species.