Among global concerns about antibiotic resistance, it is necessary to identify food-safe alternatives to enhance production. This study aimed to evaluate the impact of mannan oligosaccharides (MOS) inclusion to replace antibiotic growth promoters (AGP) in the diets of ISA Brown laying hens aged between 23 and 31 weeks. Two hundred forty hens were grouped into five treatments: Control, AGP (130 ppm of enramycin 8.00%), and 100, 200, and 400 ppm of MOS. Each treatment had 16 experimental units (each unit with n = 3) in a randomized block experimental design. Productive data (egg production %, feed intake, egg weight), egg quality variables (albumin height, yolk weight, albumin, yolk %, weight eggshell, eggshell %, equator thickness, width-pole thickness, and Haugh units), organ weights (ovary, liver, and cecum) and jejunal histomorphometry were analyzed. The egg production was not affected by the substitution of AGP by MOS. Furthermore, MOS supplementation resulted in significantly increased feed intake, larger egg weight, higher yolk weight and higher body and ovarium weight compared to the AGP group. Besides, MOS supplementation at 400 ppm demonstrated significant improvements in jejunal villus morphology indicating enhanced intestinal health. These findings highlighted the potential of MOS as an alternative to AGP, offering benefits such as improved feed intake, egg quality and intestinal health in laying hens at 400 ppm.