Nowadays, industrial poultry producers are more focused on the safety of their products, especially contaminants from feedstuffs such as mycotoxin and pesticides. The residue from animal production using antibiotic growth promoters (AGPs) may cause some problems with antimicrobial resistance in human and animals. Red yeast (Sporidiobolus pararoseus) has a cell wall consisting of β-glucan and mannan-oligosaccharides and pigments from carotenoids that may be suitable for use as a substitute for AGPs. The objective was to evaluate the effects of red yeast in laying hen diets on productive performance, egg quality, and duodenal health. A total of 22-week-old laying hens (n = 480) were divided into five groups: control diet (CON), AGP at 4.5 g/kg and red yeast supplementation at 1.0 (RY1.0), 2.0 (RY2.0) and 4.0 g/kg (RY4.0) of diet. The results show that the AGP, RY2.0, and RY4.0 groups had significantly higher final body weight compared with the other groups (p < 0.001). The red yeast supplementation improved the egg shape index (p = 0.025), Haugh unit (p < 0.001), and yolk color (p = 0.037), and decreased yolk cholesterol (p < 0.001). Diet with red yeast supplementation improved villus height to crypt depth ratio and crypt cell proliferations. In conclusion, red yeast supplementation at 2.0 g/kg of diet can substitute AGP in layer diet.