This 6-wk study was conducted to determine the effects of probiotic (Enterococcus faecium DSM 7134) supplementation of different energy and nutrient density diets on performance, egg quality, excreta microflora, excreta noxious gas emission, and serum cholesterol concentrations in laying hens. A total of 432 Hy-Line brown layers (40 wk old) were allotted into 4 dietary treatments with 2 levels of probiotic supplementation (0 or 0.01%) and 2 levels of energy (2,700 or 2,800 kcal ME/kg) and nutrient density. Weekly feed intake, egg quality, and daily egg production were determined. Eighteen layers per treatment (2 layers/replication) were bled to determine serum cholesterol concentrations at wk 3 and 6. Excreta microbial shedding of Lactobacillus, Escherichia coli, and Salmonella and noxious gas emission were determined at the end of the experiment. Hens fed the high-energy and high-nutrient-density diets had less (P < 0.01) ADFI than those fed the low-energy and low-nutrient-density diets throughout the experimental period. During wk 4 to 6 and overall, hens fed the diets supplemented with the probiotic had greater (P < 0.01) egg production, egg weight, and eggshell thickness than hens fed the diets without the probiotic. Dietary supplementation of the probiotic increased (P = 0.01) excreta Lactobacillus counts and decreased (P = 0.02) Escherichia coli counts compared with hens fed the diets without the probiotic. The excreta ammonia emission was decreased (P = 0.02) in hens fed the probiotic diets compared with hens fed the diets without the probiotic. Serum total cholesterol concentration was decreased (P < 0.01) by feeding hens with the probiotic at wk 3 and 6. Layers fed the probiotic-incorporated diets had greater (P < 0.01) high-density lipoprotein (HDL) cholesterol and lower (P = 0.03) low-density lipoprotein (LDL) cholesterol concentrations than hens fed the nonsupplemented diets at wk 6. Interactive effects (P < 0.05) of energy and nutrient density and the probiotic on excreta Lactobacillus counts and serum HDL cholesterol concentration were observed at wk 6. In conclusion, dietary supplementation of 0.01% probiotic improved egg production and egg quality and decreased excreta ammonia emission. The use of a probiotic in the high-energy and high-nutrient-density diets may be more favorable than the low-energy and low-nutrient-density diets in laying hens.