A combined pretreatment of sugarcane bagasse with glycerol and sulfuric acid was investigated based on a central composite rotatable experimental design. The following factors were varied: temperature (150–199°C), time (0.69–2.3 h), H 2 SO 4 concentration (0.0–1.1%), and glycerol concentration (55.4–79.6%). Xylans and lignin were considerably solubilized during pretreatment. Xylan solubilization, ranging between 6% and 94%, increased significantly with the increase of temperature, time, and H 2 SO 4 concentration and dropped with the increase of glycerol amount. Glycerol restricted the solubilization and full hydrolysis of xylans and the degradation of xylose. Lignin solubilization (20.6–49.4%) increased with the increase of all the experimental factors. Cellulose recovery, which was generally high, increased with the increasing of glycerol concentration and declined at high levels of the other factors. Recoveries above 97% were achieved at low H 2 SO 4 concentration and high glycerol load, whereas the lowest value (83.4%) was achieved in the longest-lasting experiment. The models based on the experimental results predicted the maximal lignin solubilization at 187.7°C, 2.3 h, 79.6% glycerol, and 0.64% H 2 SO 4 , whereas the highest yield of enzymatic hydrolysis can be expected at 194.1°C, 1.67 h, 79.6% glycerol, and 1.1% H 2 SO 4 . The optimal conditions were confirmed in control experiments. The synergistic effect of sulfuric acid and glycerol on the enzymatic hydrolysis of cellulose was demonstrated.