Systematic studies on expression and characterization of toll like receptors (TLRs) in buffalo and cattle will help to understand the innate immune response specific to each species of animals. TLR4 gene is one of the most studied innate immune receptors which is mainly triggered by lipopolysaccharide. Therefore, the present study was carried out to investigate the mRNA expression and sequence analysis of TLR4 in buffalo in comparison to that of cattle. In vitro expression of TLR4 was assessed by challenging the Peripheral Blood Mononuclear Cells (PBMC) with bacterial LPS which showed significant increase of TLR4 gene expression (P LT= 0.01) in buffaloes when compared to cattle. Further the comparison of mRNA sequence of TLR4 of buffalo and cattle revealed 96 percent similarity with the cattle sequence. The primary structure of protein showed the highest percent of leucine amino acid for both the species of animals. At the same time, the number of Leucine Rich repeats (LRR) of buffalo is varied from those present in cattle TLR4. Alpha helix is the prominent secondary structure, however, there were few amino acid variations between buffalo and cattle in certain regions of TLR4 which altered the three dimensional conformation of TLR4 proteins, this in-turn possibly can influence the binding affinity and interaction with pathogens which may influence the difference in immune response in buffalo when compared to that of cattle.