Background: Owing to the high growth rate, high protein and carbohydrate contents, and an ability to grow autotrophically, microalgal biomass is regarded as a promising feedstock for fermentative hydrogen production. However, the rigid cell wall of microalgae impedes efficient hydrolysis of the biomass, resulting in low availability of assimilable nutrients and, consequently, low hydrogen production. Therefore, pretreatment of the biomass is necessary in order to achieve higher hydrogen yield (HY). In the present study, acid-thermal pretreatment of Chlorella sp. biomass was investigated. Conditions for the pretreatment, as well as those for hydrogen production from the pretreated biomass, were optimized. Acid pretreatment was also conducted for comparison. Results: Under optimum conditions (0.75% (v/v) H₂SO₄, 160 °C, 30 min, and 40 g-biomass/L), acid-thermal pretreatment yielded 151.8 mg-reducing-sugar/g-biomass. This was around 15 times that obtained from the acid pretreatment under optimum conditions (4% (v/v) H₂SO₄, 150 min, and 40 g-biomass/L). Fermentation of the acid-thermal pretreated biomass gave 1,079 mL-H₂/L, with a HY of 54.0 mL-H₂/g-volatile-solids (VS), while only 394 mL/L and 26.3 mL-H₂/g-VS were obtained from the acid-pretreated biomass. Conclusions: Acid-thermal pretreatment was effective in solubilizing the biomass of Chlorella sp. Heat exerted synergistic effect with acid to release nutrients from the biomass. Satisfactory HY obtained with the acid-thermal pretreated biomass demonstrates that this pretreatment method was effective, and that it should be implemented to achieve high HY.