Lignocellulose, a common biopolymer in the world, is considered as one of the most important carbon sources for bioenergy production because it can be converted into various chemical compounds by biological processes. Rice straw is one of the largest quantities of lignocellulosic crop residues in the world, especially in Asian countries. The problem with using rice straw, like other lignocellulose materials, is its high resistance to enzymatic and bacterial attacks that makes a pretreatment process necessary. In this study, the potential of biological conversion of rice straw to hydrogen through anaerobic digestion (AD) process was investigated. The municipal anaerobic sludge was used as inoculumn. Heat treatment of the inoculum at 85?C for 45 minutes was employed to increase hydrogen production by inactivation of non-spore forming hydrogen consuming microorganisms and selection for hydrogen producing spore forming bacteria. Laboratory-scale experiments were carried out in completely batch bioreactors, 118 ml capacity. The mesophilic anaerobic digestions were performed and produced gases were analyzed by gas chromatography. The pretreatment process of rice straw included two chemical steps to increase the biological conversion of rice straw to hydrogen. First step was the heating of rice straw in 10% (w/w) ammonia solution for 30 and 60 minutes at 90, 120 and 150?C to remove lignin. The effect of increasing the time on alkaline pretreatment to remove lignin was investigated. The best result was observed at 120?C for 60 minutes (33.1% lignin removal). The second step was hemicellulose hydrolysis and cellulose crystallinity reduction with dilute phosphoric acid at 121?c for 60 minutes. Increasing the acid concentration leaded to increase the sugar content of liquid fraction from combined pretreatment that was easily fermentable carbon source for the initial growth of the bacteria in H2 production. Experimental results indicated that the combined pretreatment process significantly enhanced the digestibility of rice straw for anaerobic digestion to H2 production than the pretreatment methods using only ammonia solution. Combined and one-step pretreatment improved the hydrogen production yield up to 82.2% and 52.4% respectively, compared to that of the untreated one. Scanning Electron Microscopy (SEM) was used to study the morphological changes of the fiber structure of untreated and optimal alkaline and combined pretreated rice straw. The smooth, rigid and highly ordered structure of untreated rice straw, which is one of the main characteristics of the lignocelluloses, was disrupted and unsmooth after the alkali pretreatment. After the acidic pretreatment, the surface of the straw became very unsmooth and wrinkled, which is a sign that the crystal structure of the straw has been more destroyed. Moreover, FTIR spectroscopy was used to demonstrate the physical structure and changes in the functional groups of rice straw before and after more effective alkaline and combined pretreatments. The Crystallinity Index (CI) of the samples was calculated using FTIR spectra. It was 0.48, 0.42 and 0.4 for untreated, alkali-pretreated and combined-pretreated rice straw. The data showed that both treatments reduced the CI of the straw compared with untreated one that might be an important factor for improving the hydrogen yield in the bacterial digestion. Keywords: Rice straw, hydrogen production, pretreatment, anaerobic digestion