Background and Aim: Inflammation is the immune system’, s primary response to harmful stimuli. The aim of this study was to evaluate the effect of ethanolic extract of mentha pulegium on the expression of pro-inflammatory mediators of iNOS and NF-κ, B in mononuclear cells isolated from human peripheral blood activated with lipopolysaccharide. Methods: In this experimental study, mononuclear cells of 12 individuals with no history of specific disease were used. In the first group: PBS buffer was added to the culture media. Group 2: Lipopolysaccharide was used to stimulate inflammation at a concentration of 10 ng/ ml (19). Group 3: After stimulation with lipopolysaccharide, a concentration of 10 μ, g / ml ethanolic extract of mentha pulegium was used in the culture medium. Group 4: After stimulation with lipopolysaccharide, a concentration of 30 μ, g / ml ethanolic extract of mentha pulegium was used. Group 5: After stimulation with lipopolysaccharide, a concentration of 60 μ, g / ml ethanolic extract of mentha pulegium was used. Then, the expression of iNOS and NF-KB pro-inflammatory mediators in different groups wasmeasured. Data analysis was performed using SPSS software with one-way ANOVA analysis and Tukey supplementary test. Results: The results of the present study showedthat the expression of NF-κ, B gene in the second group (1. 09 ±,0. 14) significantly increased compared to the first group (P˂, 0. 05). Moreover, NF-κ, B gene expression was significantly lower in the third group (0. 48 ±,0. 09), the fourth group (0. 54 ±,0. 13), and the fifth group (0. 66 ±,0. 24) compared to the second group (P˂, 0. 05). In addition, , iNOS gene expression in the second group (1. 14 ±,0. 22) showed a significant increase compared to the first group (P˂, 0. 05). The expression of iNOS gene in the third group (0. 35 ±,0. 24) and the fourth group (0. 29 ±,0. 07) was not significantly different from the second group (P˂, 0. 05). Conclusion: The results of the present study indicated that the Ethanolic extract of mentha pulegium has anti-inflammatory effects and can be effective in altering the expression of pro-inflammatory mediators iNOS and NF-κ, B in mononuclear cells isolated from human peripheral blood activated with lipopolysaccharide.