Hydrothermal pretreatment has been proposed to enhance straw methane yield during anaerobic digestion recently. However, the combined effect of hydrothermal and organic acid pretreatment (HTOAP) needs further investigation. This study identified optimal pretreatment at 120 °C with 3 % acetic acid for 24 h by orthogonal design method. The HTOAP increased the reducing sugar content by destroying the lignocellulosic structure. A 79 % increment of methane production after HTOAP was observed compared to the untreated group. Microbial analysis showed that HTOAP enriched the relative abundance of lignocellulose-degraders, such as W5053, Thermanaerovibrio, Caldicoprobacter, as well as the syntrophic acetate oxidizing bacteria Syntrophaceticus. Moreover, Methanobacterium conducted hydrogenotrophic methanogenesis dominantly. Furthermore, the potential function analysis showed that HTOAP stimulated the expression of key enzymes in the hydrogenotrophic pathway, including carbon-monoxide dehydrogenase (EC 1.2.7.4) and coenzyme F420 hydrogenase (EC 1.12.98.1). This investigation illustrated the potential of HTOAP of rice straw to facilitate methane production.