The fermentation characteristics, structural carbohydrate degradation and enzymatic hydrolysis of rice straw ensiled with hemicellulase and Lactobacillus plantarum were examined. Fresh rice straw was ensiled in 1-L laboratory silos with no additive control (CK), L. plantarum (L), hemicellulase (HC) and hemicellulase + L. plantarum (HCL) for 6, 15, 30 and 60 days. All additives increased lactic acid concentration, and reduced pH and lignocellulosic content of the resulting silage relative to the control. The highest organic acid and residual sugar contents and lignocellulose degradation were observed in HCL silage. Hemicellulase alone or combined with L. plantarum improved the enzymatic hydrolysis with higher glucose yield and cellulose convertibility. Fresh rice straw ensiled with the combined additives increased feedstock preservation and cellulose conversion, and is thus recommended as a biological pretreatment for subsequent biofuel production.