The biodiesel production from non-edible oils has high potential as renewable and ecological fuel. Few researches have been conducted to date on the production of biodiesel from tobacco (Nicotiana tabacum) seed oil. The aim of this study was to optimize the biodiesel production from this crude oil by non catalytic supercritical methanolysis using response surface methodology (RSM). Triglyceride conversion, total and individual FAME yield, monoglyceride and diglyceride yield, and thermal decomposition degree of biodiesel were determined in the temperature and reaction time ranges of 250-350 degrees C (12-43 MPa) and 15-90 min, respectively, at a fixed methanol-to-oil molar ratio of 43:1. According to the RSM, the optimal conditions were 303.4 degrees C and 90 min, reaching a predicted maximum FAME yield of 91.1 +/- 3.2 mol%. This maximum was very close to that obtained experimentally (92.8 +/- 2.1 mol%) at 300 degrees C and 90 min. Decomposition of biodiesel became evident at 325 degrees C and 60 min of reaction due to the thermal instability of unsaturated methyl esters (methyl linoleate and oleate). The biodiesel obtained in the best experimental reaction conditions (300 degrees C and 90 min), where no thermal decomposition of FAMEs was observed, contained most of the byproduct glycerol generated, which was degraded and incorporated to the product. This biodiesel basically failed to meet the content of FAMEs as required by the standard EN 14214, the content of monoglycerides and total glycerol, and the acid value, being a little above the required upper limits. However, this biodiesel can be blended with other biodiesels of complementary features to produce a biofuel meeting the requirements of the standard EN 14214. (C) 2016 Elsevier Ltd. All rights reserved.