The intestinal microbiota is widely recognized as an integral factor in host health, metabolism, and immunity. In this study, the impact of dietary fiber sources on the intestinal microbiota and the production of short-chain fatty acids (SCFAs) was evaluated in Lohmann White laying hens. The hens were divided into four treatment groups: a control diet without fiber, a diet with wheat bran (mixed fibers), a diet with insoluble fiber (cellulose), and a diet with soluble fiber (pectin), with six replicates of four hens each. Cecal content from 24 hens was analyzed using 16 S rRNA sequencing, while SCFA concentrations were measured in blood serum. Alpha diversity analysis revealed significant variations in microbial richness and diversity among treatments, with higher species richness observed in hens fed wheat bran and cellulose, as indicated by Shannon indices. Principal Coordinates Analysis (PCoA) showed significant differences in microbial composition between the control group and the fiber-supplemented groups. The predominant phyla were Bacteroidetes, Campilobacterota, Firmicutes, and Spirochaetota, with a notable increase in Bacteroidetes in fiber-supplemented groups. Regarding SCFAs, fiber inclusion increased acetic and propionic acid concentrations compared to the control group. Diets with mixed fibers (wheat bran) resulted in the highest acetic acid levels, while propionic acid was most abundant in hens fed soluble fiber (pectin). These findings demonstrate that dietary fiber inclusion to laying hens enhances microbial diversity, stimulates SCFA production, and contributes to host metabolism and health.