Using rat gastrointestinal (GI) strips, this study investigated the stimulatory effects of methylisogermabullone (MIGB) purified from radish on the spontaneous contractility of GI smooth muscles and pharmacological mechanisms involved in the MIGB-induced GI contraction. MIGB at $30;{mu}M$ differently regulated the tone and amplitude of spontaneous GI contractility according to the region (fundus through distal colon) and orientation (longitudinal and circular) of smooth muscles: a significant increase in both tone and amplitude of spontaneous contraction in the ileum longitudinal and distal colon circular muscles and in amplitude only in the fundus, jejunum and distal colon longitudinal muscles. Pretreatment of ileum longitudinal muscles with atropine ($0.5;{mu}M$) or 4-DAMP ($0.5;{mu}M$) significantly inhibited the acetylcholine (ACh, $1;{mu}M$)- and MIGB ($30;{mu}M$)-stimulated contraction, and methoctramine ($0.5;{mu}M$) also obviously reduced the tone and amplitude increased by ACh and MIGB, respectively. In the presence of methysergide ($1;{mu}M$), pretreatment of ileum longitudinal muscles with both ondansetron ($0.1;{mu}M$) and GR113808 ($0.1;{mu}M$) significantly inhibited the contraction stimulated by 5-HT ($10;{mu}M$), but not by MIGB. Taken together, it is concluded that MIGB differently regulates the spontaneous contractility (tone and/or amplitude) of GI segments according to the region of gut and orientation of smooth muscles, and these contractile responses of GI tracts to MIGB are likely mediated, at least, by activation of acetylcholinergic $M_2$ and $M_3$ receptors.