Cardoon (Cynara cardunculus L.) is a perennial spontaneous thistle grown in Mediterranean countries and well adapted to marginal lands, recently considered as a non-food energy crop. Their seeds contain 24% of oil (dry basis). In this study, modeling and optimization of the production of fatty acid methyl esters (FAME) from cardoon oil for biodiesel uses was performed at laboratory scale, via response surface methodology, following a central composite rotatable design. FAME were obtained by transesterification of crude cardoon oil with methanol in the presence of a catalyst (sodium methoxide) for 120 min. The temperature ranged from 26 to 94 °C, the amount of sodium methoxide varied between 0.12 and 2.5 wt-% and the molar ratio methanol/oil from 0.95 : 1 to 11 : 1. The estimated yield of FAME (97%) was obtained after 30 min, at 52 °C, for a molar ratio of 6.4 : 1 and 1.4 wt-% of catalyst. In laboratory-scale model validation experiments, 94% of FAME yield was obtained after 30 min of reaction. Transesterification was performed in a 30-L reactor, under previously optimized conditions: A yield of 88% FAME was obtained after 90 min of reaction time, due to mass transfer limitations. After purification, the biodiesel showed high quality according to DIN EN 14214 standard specifications.