The in-depth research into how prehistoric human subsistence patterns responded to environmental shifts during the transcontinental spread of crops, livestock, and cultural interactions between the second and first millenium BC is a pivotal scientific endeavor that has aroused widespread attention. Northwest Yunnan, as a vital corridor for human migration, facilitated ancient human dispersals and fostered cultural exchanges. Analyzing prehistoric subsistence patterns in Yunnan, particularly in Northwest Yunnan, is crucial for understanding the dynamics and repercussions of agricultural expansion in Eurasia during prehistory. This study examined stable isotopes extracted from human bone and plant microfossils extracted from dental calculus samples collected from two sites in Northwest Yunnan. The comprehensive analysis revealed that the inhabitants of Northwest Yunnan during the first millenium BC primarily consumed C3 plants (including rice, wheat, barley, buckwheat, legumes, roots and tubers, etcetera), with C4 plants as supplements (such as millets), alongside a high intake of animal protein. With the integration of existing archaeobotanical and zooarchaeological data from neighboring sites, what emerges is a composite subsistence strategy developed by the ancestors in Northwest Yunnan during the first millenium BC, characterized by coexisting practices of planting-gathering and animal husbandry-fishing/hunting. These diversified subsistence patterns were shaped by the cultural influences from Northwest China and the local natural environment, indicating an adaptation to geographical constraints and a strategic response to climatic fluctuations during periods of low productivity.