Humans are commonly exposed to microplastics (MPs) via food. However, relatively little is known of their effect on the tissue of the gastrointestinal tract and the gut microbiome. In this study, adult male rats were fed with different concentrations (0 to 1000 μg/day) of polyethylene MPs mixed with standard pellet food for 24 days. At the end of the experiment, rats were euthanized and histopathological investigation using light and electron microscopy was performed. The gut microbiome was analyzed using MiSeq 16S rRNA amplicon sequencing. Microscopic analysis revealed a significant impact of MPs on the intestine, including visible changes to the crypt area and reduction in mucus secretion. Autophagic vacuoles were observed in the livers of the rats fed with MPs at 100 µg/day. Oxidative stress was evident in rats fed with 1000 µg/day of MPs, indicated by the presence of myeloid bodies in both intestinal and hepatic cells. The gut microbiome was also affected by MPs. Although no distinct clusters were observed in the different treatments by multidimensional scaling (NMDS). The diversity indices including operational taxonomic unit (OTU) richness and Chao1 exhibited an increasing trend with increasing MPs concentration. Pearson correlations revealed a linear increase in the relative abundance of Clostridia (R = 0.036, p = 0.003) with increasing MPs concentration. Lactobacillus faecis was the most abundant OTU in the entire dataset, and its relative abundance decreased significantly with increasing MPs concentration. This study demonstrates that MPs can disrupt cellular function and disturb microbiome balance, and in the event of prolonged exposure, organisms might experience harmful effects.