A simple hydrodynamic cavitation (HC) system was newly employed as a pretreatment method for H-2 production from cheese whey. This system offered not only the sonication effect but also an increase in the temperature of the solution. With HC pretreatment for 15 min, a higher H-2 production yield (1.89 mol H-2/mol lactose) was obtained than with other conventional pretreatment methods such as heating and sonication. HC pretreatment efficiency was synergistically increased when combined with alkaline conditions. In this way, the amount of solubilized nutrient was greatly increased and methanogens were completely removed, leading to H-2 gas of higher purity (48%) and a yield of 3.30 mol H-2/mol lactose. HC pretreatment under alkaline conditions can be used to efficiently pretreat organic-rich wastewater, including cheese whey, without any scale-up problem, and is an excellent pretreatment option for H-2 production.