Objective:To observe the influence of spent culture supernatant (SCS) of Lactobacillus acidophilus strain LA14 on the contraction of isolated intestinal smooth muscle and to discuss the related mechanism.Methods: The ileum samples of rabbits were prepared and the contraction frequency and amplitude of intestinal smooth muscle were observed as the normal control.Then the SCS, bacterium suspension, and SCS with bacterium suspension were added by an accumulative dose to the culture media (0.3 ml per times, at an interval of 6 min), respectively.Four minutes after each administration, the contractive curves were recorded for 2 min.The influences of various groups of Lactobacillus acidophilus on the contraction of isolated intestinal smooth muscle were observed.The effect of SCS on M cholinoceptor was observed by adding in order pilocarpine, atropine or SCS, and pilocarpine.Results: After continuous administration of SCS or SCS with bacterium suspension (0.6-1.5 ml), the contraction frequency of the intestinal smooth muscle was significantly lowered compared with before administration（P<0.05 or P<0.01），and there were no significant differences between before administration and other volume groups（P>0.05）.Within the range of 0.3-1.5 ml, the SCS, bacterium suspension, and SCS with bacterium suspension resulted in no significant difference in reducing the contraction amplitude, except for SCS with bacterium suspension at 1.5 ml（P<0.05）.SCS or atropine significantly inhibited pilocarpine-induced increase of contraction amplitude（P<0.05 or P< 0.01）.SCS also reduced the contraction frequency of the intestinal smooth muscle（P<0.01）.Conclusion: SCS of Lactobacillus acidophilus may inhibit the peristalsis of the intestinal smooth muscle of rabbits by blocking M cholinoceptor.