Sirenians (sea cows: manatees and dugongs) have been primary consumers in tropical and subtropical shallow-water marine ecosystems for more than 50 m.y. Though fossils of the earliest sirenians have been recovered from the Caribbean, sirenians are thought to have originated in the Tethys-Mediterranean region, which underwent significant climate change over the course of the Cenozoic. Application of stable isotope analysis can provide new insight into how diet and habitat preferences were affected by climate change and how these ecological changes correlate with diversity patterns for this region. Tooth enamel was sampled from 57 specimens of sirenians from the Tethys-Mediterranean and adjacent seas spanning the Eocene to Pliocene. Enamel δ13C values across this time interval were consistently high and indicative of diets composed mostly of marine seagrasses. In contrast, enamel δ18O values showed significant change over time and across the region. Sirenians from southern sites (i.e., Egypt, Libya) had significantly higher mean δ18O values than specimens from most European locations, suggesting variation in salinity within the Tethys-Mediterranean. The range in enamel δ18O values increased from the Paleogene through the Neogene, but enamel δ13C values remained steady, indicating that changes in environmental conditions did not necessarily coincide with major changes in dietary preferences. Declines in sirenian taxonomic diversity in the late Miocene were likely due to the rapid climate and oceanographic changes during the latter half of the Cenozoic and their impacts on available dietary resources. Elevated δ13C and δ18O values in Mediterranean sirenians during the Messinian salinity crisis corroborate the hypothesis of ecophenotypic dwarfing in these animals.