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According to IPCC Annual Report (AR-5), environmental impact assessment of any product prototype is recommended before its large-scale commercialization; however, no environmental profile analysis of any biodiesel prototype has been conducted in Pakistan. Therefore, objective of this study was to conduct a comprehensive life cycle assessment (LCA), water footprint and cumulative energy demand (CED) of biodiesel production from Jatropha curcas L. (JC) seeds oil in Pakistan. A cradle-to-gate LCA approach was applied for 400 liter (L) JC biodiesel produced in Pakistan. JC biodiesel production chain was divided into three stages i.e., 1). cultivation of JC crop 2). crude oil extraction from JC seeds and 3). crude oil conversion to biodiesel. Primary data for all the stages were acquired through questionnaire surveys, field visits and measurements in the field. Potential environmental impacts were calculated in SimaPro v.9.2 software using Eco-indicator 99 methodology. Results showed that crude oil extraction stage accounted for highest emissions (77%) to the overall environmental impact categories evaluated, followed by oil conversion stage (21%) and JC cultivation stage (02%), respectively. The three stages of JC biodiesel production chain are major contributor to ecotoxicity with a contribution of 57% to this impact category. Higher contribution to ecotoxicity was due to agrochemicals used in the JC cultivation. Similarly, fossil fuels impact category was responsible for 38% of overall environmental impacts. In addition, water footprint of JC biodiesel production chain was 2632.54 m3/reference unit. Cumulative energy required for 400L JC biodiesel production chain was 46745.70 MJ in Pakistan. Fossil diesel consumption, synthetic fertilizers use and purchased electricity were major hotspot sources to environmental burdens caused by JC biodiesel production in Pakistan. By performing sensitivity analysis at 20% reduction of the baseline values of fossil diesel used, synthetic fertilizers and purchased electricity, a marked decrease in environmental footprint was observed. It is highly recommended that use of renewable energy instead of fossil energy would provide environmental benefits such as lower greenhouse gases and other toxic emissions as compared to conventional petroleum fuels. It is also recommended that JC as a biofuel plant, has been reported to have many desired characteristics such as quick growth, easy cultivation, drought resistance, pest and insect resistance, and mainly great oil content in JC seeds (27–40%). Therefore, JC plant is highly recommended to Billion Tree Afforestation Project (BTAP) for plantation on wasteland because it has multipurpose benefits.