Abstract: Background and Objective: Nucleotide binding domain and leucine-rich repeat protein 3 (NLRP3) is reported to be involved in the pathogenesis of numerous inflammatory diseases including Alzheimer disease, Parkinson disease, Prion disease and type 2 diabetes mellitus. Previous studies have demonstrated that a stable synthetic analog of 20-hydroxyeicosatetraenoic acid (20-HETE), N-(20-hydroxyeicosa-5[Z],14[Z]-dienoyl)glycine (5,14-HEDGE), prevents vascular hyporeactivity, hypotension, tachycardia, inflammation and mortality in a rodent model of septic shock. This study was aimed to assess effect of 5,14-HEDGE on the changes in NLRP3/apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC)/pro-caspase-1 inflammasome in lipopolysaccharide (LPS)-induced septic shock in rats. Methodology: Rats were injected with saline (4 mL kg–1) or LPS (10 mg kg–1) at time 0. Blood pressure and heart rate were measured using a tail-cuff device. 5,14-HEDGE (30 mg kg–1) was administered to rats 1 h after injection of saline or LPS. The rats were sacrificed 4 h after saline or LPS injection and kidney, heart, thoracic aorta and superior mesenteric artery were isolated for measurement of caspase-1/11 p20, NLRP3, ASC and β-actin proteins as well as interleukin-1β (IL-1β) levels. Data were analysed by one-way ANOVA followed by Student-Newman-Keuls test for multiple comparisons, Kruskal-Wallis test followed by Dunns test for multiple comparisons and Student's test or Mann-Whitney U tests when appropriate. Results: Blood pressure decreased by 33 mmHg and heart rate increased by 63 bpm in the LPS-treated rats. In the LPS-treated rats, tissue protein expression of caspase-1/11 p20, NLRP3 and ASC in addition to IL-1β levels were increased. The 5,14-HEDGE prevented the LPS-induced changes. Conclusion: These findings suggest that inhibition of renal, cardiac and vascular formation/activity of NLRP3/ASC/pro-caspase-1 inflammasome involves in the protective effect of 5,14-HEDGE on LPS-induced septic shock in rats.