The trajectory and influencing factors for changes to ancient human livelihoods in the Hexi Corridor of northwest China have been intensively discussed. The Hexi Corridor is a key crossroads for trans-Eurasian exchange in both the prehistoric and historical periods. Although most studies have focused on the reconstruction of human paleodiet and plant subsistence, the diachronic change of animal utilization strategies spanning the prehistoric and historical periods remains unclear, due to the absence of zooarchaeological and isotopic studies, especially in Han Dynasty (202 BCE–220 CE). Here we report new zooarchaeological, stable isotope, and radiocarbon dating data from the Heishuiguo Cemetery of the Han Dynasty in the Hexi Corridor, indicating that humans mainly used domestic chickens, pigs and sheep as funerary objects, with other buried livestock including cattle, horses and dogs. Stable carbon and nitrogen isotope data suggest humans might have fed chickens, pigs and dogs more C4 foods (likely millets or their byproducts) than herbivorous livestock in the Heishuiguo during the Han Dynasty. Compared to other prehistoric zooarchaeological and isotopic studies in the Hexi Corridor, we detected an increasing significance of herbivorous livestock in animal utilization strategies compared with omnivorous livestock, and a basic declining weight of C4 foods in fodders from ∼2,300 to 200 BCE, which was probably induced by long-distance exchange and climate fluctuation. However, the trend was reversed during the Han Dynasty in the Hexi Corridor, primarily due to the control of the area by the Han Empire and the subsequent massive immigration from the Yellow River valley of north China.