Abstract To understand the resource and environmental costs of bioethanol production, this study estimates the water and farmland use and CO2 emissions in China due to first- and second-generation bioethanol production technologies using a multi-regional input-output-based hybrid life cycle assessment model. Sensitivity analysis and structural path analysis are combined to investigate the key pathways for bioethanol production impact mitigation. Results show that the first-generation technology has higher resource-use and environmental impacts compared to the second-generation technology. The gasoline-to-bioethanol transition enables CO2 emission reductions, but at the cost of increased water and farmland use. Key impact mitigation pathways are then investigated for the first four production layers (PL0→3) in bioethanol industries because these layers contribute to a significant environmental footprint. For PL0, water-saving technologies must be developed to reduce direct water use in first-generation bioethanol production. For PL1, the primary production material suppliers of the bioethanol industry, such as biomass feedstock, food and tobacco, and water supply industries were ascertained with high potential for impact mitigation. For PL2→3, key pathways are investigated by combining their sensitivity and impact values. The results highlight the significance and necessity of cross-sectoral collaboration for resource and environmental impact reduction.