Abstract: Background and Objectives: Alpha-linolenic acid which is one of polyunsaturated fatty acids is very important for the continuity of human life. Alpha-linolenic acid is a protective food for the heart and cardiovascular system. In addition, this substance is a powerful anti-inflammatory agent. Increases in inflammatory agents are accompanied cystitis induced by lipopolysaccharide (LPS). Therefore, this study aimed to determine whether alpha-linolenic acid, which has anti-inflammatory properties, is protective effect against LPS-induced cystitis. Materials and Methods: Cystitis were induced by intravesical LPS application in mice and treated with alpha-linolenic than cyclooxygenase-2, phospholipase A2 and inducible nitric oxide synthase (iNOS) enzymes were analyzed by Enzyme-Linked Immuno Sorbent Assay (ELISA) method in their bladder IL-6 and TNF-α in their serum. Differences in results between tissues were tested by one way analysis of variance (ANOVA) corrected by Bonferroni multiple-comparison. Results: While cystitis increased expression of IL-6, TNF-α, NF-κB, cyclooxygenase-2, phospholipase A2 and iNOS enzymes the application of alpha-linolenic acid reversed this increase. Conclusion: This study discovered the alpha-linolenic acid that can be beneficial for LPS-induced bladder inflammation. This study will help the researcher to uncover the critical areas of bladder inflammation caused by Gram negative bacteria.