Piperine is one of the alkaloid compound which contained in the piper (Piper nigrum Linn). Alkaloid compound has been studied to bond specifically at AChM3 receptors. The airway constriction process is caused by smooth muscle contraction of the respiratory tract. It is occurred due to the influence of the muscarinic acetylcholine 3 (AChM3) binding to the G protein that resulting in an increase of Ca2+ intracellular. The purpose of this study determined the effect of piperine alkaloids on acetylcholine receptors toward the tracheal smooth muscle contraction activity. The study implemented in-vitro method which is use organ bath apparatus and isolated guainea pig trachea. Krebs solution is used to subtitude physiological buffer. Acetyl-β-Methylcholine agonist was used as a trigger to contract guinea pig trachea smooth muscle. Acetyl-β-Methylcholine is given gradually from the concentration of 2x10-8 M to 2x10-2 M. Furthermore, piperine was used at the dose 10 μM and 50 μM. Data was analyzed in the form of curve which depict the correlation between the contraction response percentage and agonist concentration. In addition from the curve pD2 values also can be obtained. Statistical analysis by using one way ANOVA with 95% confidence level. The results showed that piperine dose at 10 μM and 50 μM was able to shift the curves response percentage contraction with agonist concentration to the right. Piperine at the 10 μM and 50 μM could not restore the contraction response to 100% of Emax. The trachea which given 10 μM piperine could reach maximum response values at 68.49% contraction. Meanwhile that given 50 μM piperine only has 70.28% contration. pD2 value of control, piperine 10 μM, piperine 50 μM group was 5.50; 4.67; 4.41 respectively. Overall, from the research above, it can be conclude that the piperine has role as noncompetitive antagonist at the muscarinic acetylcholine receptor 3 (AChM3).