Acid and alkaline pretreatments for enhanced hydrogen production from cassava stillage were investigated in the present study. The result showed that acid pretreatment was suitable for enhancement of soluble carbohydrate while alkaline pretreatment stimulated more soluble total organic carbon production from cassava stillage. Acid pretreatment thereby has higher capacity to promote hydrogen production compared with alkaline pretreatment. Effects of pretreatment temperature, time and acid concentration on hydrogen production were also revealed by response surface methodology. The results showed that the increase of all factors increased the soluble carbohydrate production, whereas hydrogen production was inhibited when the factors exceeded their optimal values. The optimal conditions for hydrogen production were pretreatment temperature 89.5 °C, concentration 1.4% and time 69 min for the highest hydrogen production of 434 mL, 67% higher than raw cassava stillage.