Abstract The excavations in Biśnik Cave created an opportunity to investigate changes in δ13C and δ18O values of Ursus spelaeus teeth from one site along a 200,000 year period of sedimentation, for the first time in Eastern Europe. Biśnik Cave (Kraków–Częstochowa Upland, southern Poland) is a multilayered archaeological and paleontological site with late Middle Pleistocene and Late Pleistocene sediments, where several important changes of Quaternary climate have been recorded. The project considered if and how detailed the isotopic data from one multilayered site record these great climatic changes. The method used was an isotopic analysis of carbonate in bioapatite from tooth enamel. Teeth of cave bear (U. spelaeus) were chosen as research material. The results showed that the δ18O values of cave bear teeth from Biśnik Cave range from −14.9 to −4.4‰ VPDB with some variation between particular layers. Values of δ13C vary between −20.5 and −14.5‰ VPDB. The analysis revealed that isotopic record is diverse between different types of teeth, probably due to differences in time of growth and different impact of nursing and hibernation. The results verify the responsiveness of this species to great climatic changes during Middle and Late Pleistocene in Eastern Europe. Cave bear was an ecologically inflexible species, associated with the same type of food during 200,000 years and not able to cope with the coldest phases of the Pleistocene.