Abstract: The purpose of this study was to investigate the effect of probiotic (0, 400, 1000 and 2000 g Bioplus 2B ton-1 feed providing 0, 1.28 x 106, 3.2 x 106 and 4.6 x 106 cfu g-1 feed concentration) in different levels of barley substitution for corn diets (0, 50 and 100%) on laying hen`s performance, egg quality, blood factors and histological changes in duodenum. Evaluated traits were egg production, egg weight, egg mass, feed consumption, feed conversion ratio, shell thickness, shell hardness, Haugh unit, egg cholesterol, plasma cholesterol, plasma triglyceride and histological changes of duodenum. Using different levels of substitution of barley for corn showed highly significant decrease (p<0.01) in feed consumption and feed conversion ratio, highly significant increase (p<0.01) in goblet cell numbers and epithelium surface folds of villus, significant increase (p<0.05) in egg weight and damaged apical cells of villus, without any effect on other traits. Although, using the different levels of probiotic caused highly significant increase (p<0.01) in goblet cell numbers, significant increase (p<0.05) in feed consumption, feed conversion ratio and destroying apical cells of villus and significant decrease (p<0.05) in plasma cholesterol and triglyceride, but it had no effects on other traits. Consumption of probiotic alone had no effect on egg cholesterol (mg g-1 of yolk), but caused significant decrease (p<0.05) in egg cholesterol (mg g-1 of yolk) among fourth level of probiotic and another levels, in corn based diets. Interaction of different percentages of substitution of barley for corn and probiotic levels had highly significant effect (p<0.01) on feed consumption, feed conversion ratio and egg cholesterol (mg g-1 of yolk) and significant effect (p<0.05) on Hough unit.