Introduction: The function of acetylcholinesterase accelerates the acetylcholine hydrolysis process and protects the hemostasis of this neurotransmitter in the central nervous system and the peripheral tissues. Due to the fact that in diseases, such as Alzheimer's disease, the level of acetylcholine is reduced, the administration of acetylcholinesterase inhibitor drugs (AChEID) is one of the methods for preventing the progression of this disease. The majority of the researchers attempted to investigate drugs with lower side effects, especially from herbal sources. The aim of this study was to investigate the effect of methanol extract of Mentha pulegium leaves on the enzyme activity of AChE. Materials & Methods: In this study the AChE enzyme was partially isolated from the bovine brain using homogenization, centrifugation, ammonium sulfate precipitation, and dialysis at 4 º C. The rate of production of thiocholine from acetylthiocholine iodide was measured according to Ellman's method. Subsequently, the methanolic extract of Mentha pulegium leaves was prepared by the maceration method. Then the inhibitory effect of the extract on the AChE was measured. Ethics code: IR. umz. rec 1397. 090 Findings: The total activity and specific activity of the partially purified AChE were determined as 1143. 78 U and 4. 10 U/mg, respectively. The optimum pH and temperature of the acetylcholinesterase were determined to be 7. 5 and 45° C respectively. Moreover, the Km and Vmax of this enzyme were estimated at 6. 49 mM and 59. 88 mM/min. Discussion & Conclusions: The results of this study showed that the methanolic extract with a concentration of 26. 66 mg/ml had significant effect on the activity of this enzyme. According to the results, this extract can inhibit the AchE in form of mixed inhibition. Therefore, Mentha pulegium leaf could be considered by the researchers in study and investigation of pharmaceutics.