Background: Structure and functions of different organs especially cardiovascular, cerebrovascular and reproductive systems are compromised by the consumption of high fat diet. Gastrointestinal system has gained less attention in this respect. An intact and efficient gastrointestinal barrier protects us from different harmful agents. Objective: Our objective was to observe the effect of high fat diet on the uptake of particles by the peyer's patches (PP) and its transport to mesenteric lymph nodes of rats. Materials and Methods: Twenty, four weeks old male Wistar rats were used for the study. Rats were divided into two groups, control (n = 10) and HFD (n = 10) group. They received the respective diet for 6 weeks. 1 hour prior to sacrifice, 1 ml of fluorescent latex bead solution was given to all rats by gavage tube. After sacrifice, small intestinal samples containing PP were collected. Samples of ileum were fixed in formalin, cut in microtome for hematoxilin and eosin stain to observe and analyze small intestinal morphology. Ileum samples containing Peyer's patches and mesenteric lymph nodes were frozen in liquid nitrogen, cut in cryostat, taken on poly-l-lysine coated glass slides, observed and analyzed the number of fluorescent particles under confocal laser scan microscope. Statistical comparisons were performed by student's independent t test. Result: Body weight of the rats were significantly higher in HFD group as compared to the control group (C = 223 ± 25.6 gm, HFD = 339 ± 11 gm, p < 0.001). Height of the villi and crypt depth were significantly higher in HFD group as compared to the control group (C = 217 ± 7.7 μm, HFD = 324 ± 54.3 μm, p < 0.001 and 108 ± 7.6 μm, HFD = 136 ± 31.4 μm respectively). Uptake of particles by the PP was significantly higher in HFD group as compared to the control group (C = 23 ± 7.5, HFD = 106 ± 25.4, p < 0.001). Number of particles in the mesenteric lymph nodes was not different significantly between the two groups (C = 8 ± 3, HFD = 12 ± 3). Conclusion: We conclude that HFD for 6 weeks in rats increased the uptake of particles by the PP. We recommend that further studies should be conducted to find out the underlying mechanism to prevent or ameliorate such changes by several modulating agents. © 2013 Japan International Cultural Exchange Foundation & Japan Health Sciences University.