The present study was conducted to investigate the effect of dietary supplementation of mannan-oligosaccharide (MOS) on performance, immunological responses, nutrient digestibility and ileal microbial populations in laying hens. A total of 150 Hy-line W-36 Leghorn hens of 55-wk of age were randomly distributed among 6 replicate cages of 5 experimental diets. Dietary treatments consisted of five different levels of MOS (0, 0.05, 0.10, 0.15 and 0.20%), that fed during a 77d feeding trail including 7 days for adaptation period and 70 days as main recording period. Hen-day egg production and egg weights were recorded daily, but all performance parameters were reported as two 35 days intervals. The results showed that different MOS levels had no effect on egg weight and feed intake. In contrast to second 35 d period, dietary MOS supplementation had a significant (P 0.05) effect on feed conversion ratio (FCR), with the best FCR values assigned to the birds on 0.10 and 0.15% MOS-diets. Dietary supplementation of MOS caused a considerable increase in egg production (P 0.01) and egg mass (P 0.05). The highest egg production and egg mass assigned to the hens fed on diets contain in 0.10 and 0.15% MOS levels. Although internal and external egg quality measurements weren¢t influenced by experimental diets during both sampling periods, but there was a slight improvement in Haugh unit in second 35 d period. The primary antibody titer against sheep red blood cell (SRBC) increased (P 0.05) by dietary MOS supplementation; however, dietary MOS inclusion had no effect on antibody production against Newcastle disease virus and SRBC during second response. Although serum closterol concentration wasn¢t affected by dietary treatments, however, dietary supplementation with MOS caused a considerable decrease in serum LDL (P 0.001) and triglicerides (P 0.01) and increase in HDL (P 0.01) concentrations, with the lowest TG assigned to 0.15% MOS-diets and the highest HDL assigned to 0.10% MOS level. On the other hand, incremental MOS levels up to 0.20% decreased LDL. The results of nutrient digestibility showed that the dietary MOS supplementation caused a marked improvement in dry mater (P 0.05), ether extract and crude protein (P 0.01) digestibilities. The best nutrient digestibility assigned to the birds on 0.10 and 0.15% MOS-diets. Dietary treatments had considerable (P 0.05) impact on ileal microbial populations. Although MOS supplementation as compared with control group, had no effect on total ileal bacterial count and E. coli, but caused a significant (P 0.01) decrease in Salmonella and increase in Lactobacillus. The present findings indicate that MOS supplementation of laying diets could improve performance, blood biochemical parameters and nutrient digestibility, with a part of these beneficial effects may be due to modulation of intestinal microbial populations. Keywords : laying hen, mannan-oligosaccharide, performance, blood biochemical parameters, nutrient digestibility, ileal microbial population