For the use of biologically produced H2, removal of CO2 is an indispensable process. Unlike conventional CO2 removal methods, this study proposed a self-generated high-pressure dark fermentation (HPDF) process as a novel strategy for directly producing high-calorific bio-H2. The pressure was automatically increased by self-generated gas, while the maximum pressure inside fermenter was restricted to 1, 3, 5, 7, and 10 bar in a batch operation. As the pressure increased from 1 to 10 bar, the H2 content increased from 55% to 80%, whereas the H2 yield decreased from 1.5 to 0.9 mol H2/mol hexoseadded. The highest H2 content of 80% was obtained at both of 7 and 10 bars. Increased lactate production with increased abundance of lactic acid bacteria was observed at high-pressure. Despite the lower H2 yields at high-pressure conditions, HPDF was found to be economically beneficial for obtaining high-calorific bio-H2 owing to the low CO2 removal cost.