In this work the influence of milling on clean depithed sugar cane bagasse (as a pretreatment step) on two stage acid hydrolysis was evaluated. Two different depithed bagasse samples were studied, very fine powder that passed mesh No 325 and coarse bagasse without milling (3 cm fiber bundle length). The effect of bagasse particle size on parameters was studied as well as hydrolysis of the two particle sizes were carried out. In the present study, the bagasse material was analyzed by scanning and transmission electron microscopy (SEM and TEM) which confirmed that the particle size after the milling process was decreased to the nanometer scale form (20-50nm) that catalyzed the hydrolysis process. Hydrolysis time, temperature and liquid to solid ratio were kept constant at 30 min., 100 oC and 10:1 respectively, while concentrations of sulfuric acid were varied. In the first stage the concentration of acid ranged between 2% to 10% (wt. / wt.) and in the second stage it ranged between 10% to 30 %( wt /wt.). The results revealed that on using fine powder bagasse, in the first acid hydrolysis stage, the total dissolved sugars were 2 to 1.3 times more than that on using coarse bagasse, based on acid concentrations. Concerning concentration of xylose in the hydrolyzed solution, increasing acid concentration from 2% to 10%, on using coarse bagasse, xylose concentration was increased from 4 to 9.9 mg/L while on using fine powder bagasse it increased from 7.5 to 12.9 mg /ml. In case of second stage, on using fine powder bagasse the total dissolved sugars were 1.7 times more than on using coarse bagasse. Concerning concentration of xylose in the hydrolyzed solution, on increasing the acid concentration from 10% to 30%, the increase was from 0.7 to 1.8 mg / ml on using coarse bagasse, while on using fine powder bagasse the increase was from 1 to 2.3 mg /ml. The higher efficiency of fine powder bagasse may be attributed to that; the mechanical action can promote degradation and damage of lignocellulose internal structure and destroy the crystalline structure of cellulose, in addition generation of higher surface area of the biomass particles. Such actions increase accessibility and mass transfer of acid which lead to higher hydrolysis of hemicellulose and cellulose.