In this paper, we apply standard zooarchaeological methods and novel osteological approaches to analyse faunal remains from five Middle–Late Holocene sites in the southern Tibetan Plateau (STP). Framed by direct radiocarbon dates on taxonomically classified bioarchaeological remains and compared with published palaeoclimate data, our findings revealed a three-stage process of agro-pastoral development in the STP ca. 5.5 to 1.0 ka. In the first phase, habitation was restricted to the lower southeastern part of the plateau and human subsistence essentially based on foraging and low-level pig–millet farming. With the onset of colder and drier climatic conditions ca. 3.8 ka, the study area witnessed a growing human presence at higher elevations in its central and western parts, together with a shift towards bovid husbandry and barley cultivation, that is, agricultural practices that originated in west Asia; these were likely introduced to the STP following the eastern margin of the TP and/or arrived by sub-Himalayan transfer. Climate and ecological degradation might have contributed to the decline of local game in favour of cold-and-dry-tolerant pastoral livestock and crops. Our work shows that Middle–Late Holocene climate change, ecological change, human subsistence shifts, and prehistoric cultural transmissions are intimately connected.