Object: To compare the inhibitory effects of loperamide on spontaneous and acetylcholine induced contractions of isolated ileum of Guinea pig. Study Design: Comparative controlled in-vitro experimental Study. Place and Duration of Study: This study was conducted at Department of Pharmacology, Basic Medical Sciences Institute (BMSI), Jinnah Postgraduate Medical Center (JPMC) Karachi for the period of six months. Materials and Methods: Isolated small intestine of twenty four Guinea pigs was used and fresh Tyrode nutritional solution was prepared for each experiment. They were divided in three groups. In group I, standard concentration of acetylcholine was detected. While in group II ileal spontaneously contractions and in group III acetylcholine primed ileum were exposed to serial dilutions of loperamide (from 10 -18 to 10 -3 gm/ml) and effects were observed and recorded by 7B Grass Polygraph. Results: Loperamide inhibits the frequency of spontaneous contractions of ileum ranging from 13.17% to 25.36% and amplitude ranging from 19.74% to 37.74% with different serial dilutions. While loperamide inhibits the frequency of acetylcholine induced contractions of ileum ranging from 25.32% to 39.89% and amplitude ranging from 32.38% to 45.27% with different serial dilutions. Statistically: We also found significantly (p<0.05) difference observations in between group II and III, due to higher inhibitory values in group III. Conclusion: Loperamide was found more potent inhibitor for acetylcholine induced contractions than for the spontaneous contractions of isolated ileum of Guinea pig.