Background: bacteriocins are heterogeneous inhibitory substances that could affect the bacteria belonging to the same genus. Both gram-positive and gram-negative bacteria produce bacteriocins. One of the best sources of producing bacteriocins is Lactobacillus. The aim of this study was to isolate and purify bacteriocin from Lactobacillus rhamnosus and assess its effects on Pseudomonas aeruginosa and synthesis of its lipopolysaccharide. Methods: L. rhamnosus was prepared and cultured at MRS broth and incubated at 37º C for 24 hours. Then, the medium was centrifuged for the isolation of bacteriocin and the supernatant was considered as bacteriocin. Antibacterial properties of different concentrations of bacteriocin (50, 100, 200, and 400 μ g/mL) against P. aeruginosa were assayed by using agar diffusion and broth micro dilution methods. Also, the effect of bacteriocin against lipopolysaccharide synthesis in P. aeruginosa was analyzed by using one unit of minimum inhibitory concentration (MIC) for bacteriocin. Results: The results showed that all bacteriocin concentrations had antibacterial activity against P. aeruginosa. The MIC value was 31. 25 μ g/mL and minimal bactericidal concentration (MBC) was 62. 5 μ g/mL. Also, the synthesis of lipopolysaccharide decreased during P. aeruginosa growth period, and it reached zero after 5 hours. Conclusions: The results of this study showed the antibacterial effect of bacteriocin isolated from L. rhamnosus against P. aeruginosa. In addition, this bacteriocin prevented the lipopolysaccharide synthesis in P. aeruginosa.