The study explores the potential of the Croton macrostachyus seed, a non-edible feedstock found in abundance in the southeastern parts of Ethiopia, for the production of biodiesel. Soxhlet extractor was used to extract, 53.34% of the oil from the Croton macrostachyus seed. This oil was further purified and the resulting product was known as the purified Croton macrostachyus seed (P-CMS) oil. After obtaining the P-CMS oil, biodiesel was produced by the transesterification process. Focus was made on the optimization of biodiesel production by varying the identified process parameters, through the Box- Behnken Design using response surface methodology (RSM) (Design-Expert (R) Version 11 software). The crude biodiesel was purified, characterized and complied with the ASTM D6751, United States and the EN 14214, European Union standards. The optimum conversion efficiency of the P-CMS oil to fatty acid methyl ether (FAME) was 96% at the optimal conditions (6:1, methanol to oil ratio; 1%, catalyst loading; 50 degrees C, reaction temperature). A plan for the environmental impact assessment and life cycle assessment were provided to evaluate the sustainability of this new feedstock and the biodiesel produced from it. Results of these studies indicated the prospective of Croton macrostachyus seed as a new biodiesel feedstock. (C) 2020 Elsevier Ltd. All rights reserved.