In the present work, a combined (acid + thermal) pretreatment was applied for the enhanced fermentative H2 production (FHP) from Laminaria japonica. Various pretreatment conditions including HCl concentrations, heating temperatures, and reaction times were optimized via response surface methodology (RSM) with a Box-Behnken design (BBD). Through regression analysis, it was found that H2 yield was well fitted by a quadratic polynomial equation (R2 = 0.97), and the HCl concentration was the most significant factor influencing FHP. The desirable pretreatment conditions found were HCl concentration of 4.8%, temperature of 93 °C, and reaction time of 23 min, under which H2 yield reached to 159.6 mL H2/g dry cell weight (dcw). The main organic acids produced were acetic and butyric acids that related closely with H2 production. The concentration of hydroxymethylfurfural (HMF), a byproduct formed during the pretreatment process, showed an inverse relationship with H2 yield, indicating that pretreatment conditions for the H2 production from L. japonica were successfully optimized, by increasing the hydrolysis rate of the feedstock and also reducing the formation of HMF.Highlights► This study presents the optimization of combined (acid + thermal) pretreatment for the enhanced fermentative H2 production from Laminaria japonica using response surface methodology (RSM) with a Box-Behnken Design (BBD). ► HCl concentration (0-12%), thermal pretreatment temperature (60-160 °C), and reaction time (5-40 min) were chosen as three independent variables. ► There was an inverse relationship between H2 yield and produced hydroxymethylfurfural (HMF) concentration with a R2 value of 0.84.