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Prevalence of obesity is increasing worldwide. One of the major risk factors for obesity is consumption of high fat diet (HFD). Gastrointestinal tract (GIT) is the first organ where HFD comes in contact with the body. But, the effect of HFD on the GIT especially the GIT barrier is not investigated properly. M cells are present in the Follicle associated epithelium (FAE) of Peyer’s patches (PP) of GIT and are important component of intestinal barrier. Healthy and adequate number of M cells is important for an effective intestinal barrier. Intestinal tight junction protein Claudin 4, situated in between the enterocytes of Peyer’s patches (PP), regulates the permeability through the intestinal mucosa. Reduced Claudin 4 is responsible for increased paracellular transport of antigenic materials. Calprotectin is an inflammatory marker secreted by neutrophils during inflammation. Its level is considered specific for intestinal inflammation. The objectives of this study were to investigate the expressions of M cells and Claudin 4 in the PP and to determine the fecal calprotectin level (FCP) of male Wistar rats fed HFD. Four weeks old, twenty male Wistar rats were divided into chow (n=10) and HFD (n=10) groups. After 6 weeks of consuming the respective diets, stool and GIT segments containing PP were collected. After tissue processing, tissues were sectioned into 3 micrometer thickness and were taken on poly-L-lysine coated glass slides. Immunohistochemical staining was done by rat M cell specific CK-8 antibody and anti-Claudin 4 antibody. Scoring was done to calculate the average number of M cells and Claudin 4 in the PP of both groups under light microscope. FCP were measured using a commercial enzyme linked immunoassay (ELISA) kit. Statistical analysis was done by chi-square test and independent T-test. Data are presented as mean ± SD. A p value