The main alkaloid found in white pepper (Piper nigrum L.) is piperine. Piperine is one of the most studied compound and has promising effect that isolated from Piper nigrum L. Piperin was reported may inhibit the release of histamine from mast cells by inhibiting the signal pathway mediated by IgE. Based on the fact, piperin was expected to has the effect of antagonism action at the histamine receptor. The aim of this study was to extracting alkaloid compound from Piper nigrum L. and investigate its effects on H1 receptor. This research was conducted by investigated the effect of alkaloid of pepper on the guinea pig ileum smooth muscle contraction induced by histamine. Alkaloid was obtained from the pepper extract and then identification of piperin was done by using TLC, FTIR and UV-Vis spectrophotometry and melting point assay. The alkaloid was administered at the doses of 1000 μM and 5000 μM. On this in vitro study, the type of antagonism and reversibility will be investigated. In this study was also conducted in silico study of piperin using the AutoDock software. The result showed that alkaloid of pepper containing piperine based on the result of the FTIR, UV-Vis spectrophotometry, and melting point assay. Alkaloid also able to inhibit the contraction of isolated guinea pig ileum smooth muscle induced by histamine. The pD2 values of H1 receptor shifted significantly on the dose of 5000 μM (p < 0.05) with the types of non-competitive antagonists observed from the shape of the contraction response curve that was not reach 100% Emax. Reversibility assay showed that by replaced buffer tyrode every 5 minutes for 30 minutes , the bond of alkaloid to the receptor able to dissociate. In the in silico study, piperine was observed can be bind to receptors H1 (docking score :-5.70). Piperine bound at amino acid Lys179 which is one of the important proteins in the histaminergic activity. The conclusion of this research is the alkaloid of pepper containing piperine and has the activity as a non-competitive antagonist at the H1 receptor.