Fine particulate matter with a diameter ≤2.5 μm (PM_2.5) pollution poses a global public health crisis, demonstrating significant threats to human health. This study focused on the strategically important Chengdu-Chongqing Economic Circle in western China, systematically comparing the toxic effects of urban and rural PM_2.5 across five levels. PMF and regression analysis were used to identify source contributions, dual-omics to pinpoint key molecules, and epidemiological data with a GAM model to assess health risks. Findings demonstrate that rural PM_2.5 possesses greater biotoxicity than its urban counterpart. Cytotoxicity in urban and rural PM_2.5 originated from road dust/vehicle emissions and biomass burning, respectively. Subsequently, integrated omics and molecular biology analyses identify kinesin family member 20A (KIF20A) as a shared key target, which mediates toxicity induced by both urban and rural PM_2.5. Finally, epidemiological analysis reveals that females and ≥65 years old exhibit relatively high sensitivity to urban PM_2.5 exposure trends, with rhinitis showing a comparatively higher impact among various related diseases. The novelty of this work lies in its pioneering application of a multi-tiered investigative approach. This approach spans “environmental samples-cellular mechanisms-population health” within the Chengdu-Chongqing economic circle context, systematically elucidating common and distinct respiratory health risk of urban and rural PM_2.5. This work offers a vital scientific foundation for advancing region-specific, precise air pollution prevention and control measures.