The objective of this study was to study the effect of probiotic supplementation in diet on egg production, egg quality, intestinal morphology, and bacterial population in caecum of laying hens. A total of 768 Lohmann Brown-Classic laying hens, aged 42 - 54 weeks, were used. The laying hens were divided into 3 groups, each with 8 subgroups of 32 birds each. The experiment was planned to use a completely randomized design method. The 3 experimental groups were classified based on their dietary treatment as follows: 1) Basal diet (without probiotic supplementation), 2) Basal diet with probiotic 0.01 and 0.02 % in 3, respectively. Data was collected for 12 weeks. Adding probiotic in the diet was no significant difference on egg production, egg quality, and bacterial population in caecum. However, probiotic supplementation in the diet increased villi height of the duodenum significantly improved (p < 0.02), villi height per depth crypt of the duodenum significantly improved (p < 0.05), villi height per depth crypt of the jejunum significantly improved (p < 0.01), and villi height per depth crypt of the ileum significantly improved (p < 0.02). Therefore, studies have shown that dietary probiotic supplementation can improved small intestinal morphology are the most important indexes for measuring the digestive and absorption function of the small intestine are more efficient. HIGHLIGHTS Probiotic (Bacillus subtilis) is a gram-positive, non-pathogenic bacterium that can inhibit digestive enzymes such as protease, amylase, and lipase. These enzymes help increase the efficiency of digestion and absorption of nutrients. As a result of the working mechanism of Bacillus subtilis, it helps strengthen the intestinal wall, eliminates pathogenic microorganisms, reduces damaged intestinal mucosal cells, and improves intestinal absorption, resulting in increased production. Effect of probiotic (Bacillus subtilis) supplementation in laying hen diets on total bacterial load in appendix. No difference was found in the load of total bacteria and Escherichia coli (P > 0.05). Its beneficial properties stimulate the growth of beneficial bacteria in the intestines, such as Bifidobacteria sp. and Lactobacillus sp., causing the production of lactic acid, which makes the colon more acidic, resulting in inhibition of the growth of pathogenic microorganisms. GRAPHICAL ABSTRACT