Background and aim Atractylodes japonica Koidz (Compositae) has been commonly used to treat the gastrointestinal (GI) disorders in Korean traditional medicine, but its pharmacological roles in the regulation of GI motility have not been clarified yet. Methods Atractylodes japonica was sequentially partitioned with MeOH, n -hexane, CHCl 3 , EtOAc and n -BuOH saturated with H 2 O, and the effects of Atractylodes japonica extracts on the spontaneous contractility of GI muscle strips prepared from rats were measured. Results Among five different fractionations, EtOAc extracts of Atractylodes japonica (AJEA) dose-dependently increased the low frequency contraction of distal colon longitudinal muscles (DCLM), and the ED 50 values were revealed to be 1.71 × 10 −9 g/ml. Among GI tracts, a prominent contractile response to AJEA was observed only in the DCLM. The contractile patterns produced by AJEA remarkably differed from those caused by acetylcholine and 5-HT. 4-DAMP and methoctramine at 0.5 μM significantly blocked the AJEA (1.0 μg/ml)-induced contraction of DCLM, but ondansetron, GR113808 and methysergide at 1.0 μM in combination did not change the AJEA-induced DCLM contractions. Acetylethylcholine mustard (5.0 μM) significantly diminished the AJEA-induced DCLM contractions, whereas p -chlorophenyl alanine (1.0 μM) did not affect the stimulatory effects of AJEA on the DCLM contractions. Conclusion The present results suggest that AJEA may specifically act on the DCLM among GI smooth muscles, and AJEA-induced DCLM contraction is likely mediated, at least, by activation of ChAT and acetylcholinergic muscarinic receptors.