This study investigated the individual and interactive effects of three factors - temperature, inoculum/substrate ratio (ISR) and inoculum typology - on the anaerobic digestion of corn ethanol distillery wastewater. Biochemical methane potential assays planned with factorial design with two independent quantitative variables on three levels (ISR: 1:1, 2:1 and 3:1; temperature: 30°C, 33.5°C, 37°C) and one independent qualitative variable (inoculum type: suspended, granular, mixed) have been performed. Response Surface Methodology has been used to study the effect of the factors with the aim of maximizing the specific methane yields (YCH4) obtainable with this substrate. The results show that all three investigated factors influence in a significant matter the YCH4, the ISR having the strongest effect on it. The temperature has significant influence on the YCH4 only in combination with high ISR values. The optimal conditions for the maximum YCH4 (551 mL CH4 g−1 VSadded) have been found at 37°C operating temperature, ISR=3:1 and using granular inoculum. These conditions gave rise to a 4-fold increase of YCH4 with respect to the worst combination of factors (YCH4=129 mL g−1 VSadded for the suspended inoculum type, at 30°C and ISR=1:1). The results improve the knowledge on the digestion of this substrate, providing information for successful process up-scaling.