BACKGROUND: Barley cultivars contain different anti-nutritional factors that have negative effects on the intestinal ecosystem and reduce blood cholesterol concentration. Objectives: This study aimed to determine the effect of processing barley cultivars on the intestinal morphology, enzyme activity and volatile fatty acids of the small intestine and serum lipid levels of broiler chickens. Methods: The experiment was carried out using a factorial experiment with a completely randomized design. Factors were barley cultivars (Fajr, Reyhaneh, and Yosef), diet form (mash or pellet) and enzyme level (0 and 0.05 g/kg dry matter). Around 927 one day of old Ross 308 broiler chickens were assigned into 12 treatments, 3 replicates and 27 chickens in each replicate. Results: Pancreatic amylase, lipase enzyme activity and digesta pH are affected by main (except for cultivar) and interaction effects (P< 0.05). Feed Processing (pelleting, adding enzyme or combination of pelleting and enzyme) of barley cultivars, especially diets containing Ryhaneh barley cultivar reduced digesta viscosity and increased the concentration of volatile fatty acids and relative percentage of acetic acid and butyric acid (P<0.05). Diets containing Fajr barley cultivars, enzyme or pelleted diets significantly decreased blood glucose, cholesterol and HDL concentration (P<0.05). Conclusions: Due to lower amount of soluble NSP compared with the other barley cultivars, Ryhaneh cultivar resulted in a better response to processing as well as enzyme addition, and improved the morphological characteristics of the small intestine