Objective：The aim of the study was to explore the effect and possible mechanism of 17 β-estradiol（E2）on the expression of small conductance Ca2+ activated K+ channel 3（SK3）in rat colonic smooth muscle. Method：24 male Sprague-Dawley（SD）rats were randomly divided into 4 groups. Silicone tubes containing different solution was subcutaneously implanted in different groups of rats. Group C was filled with the solvent；Group EP was E2 in corn oil（0.3 mg/mL）which can keep serum levels of E2 at physiological levels；Group ES was E2 in corn oil（0.9 mg/mL）which can keep serum levels of E2 at supraphysiologic levels；Group EI was ICI 182780（estrogen receptor antagonist）plus E2（0.3 mg/mL）. The contraction of muscle strips and SK3 expression in colonic smooth muscles were detected at 14 days after tube implanted. SK3 were overexpressed in SMCs and Ca2+ concentration in cells were detected by laser scanning confocal microscope. Results：The contraction of muscle strips of EP group and ES group is weaker than other groups（P < 0.05）after Cch stimulating. And the SK3 expression was upregulated in EP and ES groups（P < 0.05）. Ca2+ decreased in SMCs after Cch stimulating in SK3 overexpression group and E2 stimulating group compared with control group. Conclusion：These findings indicate that E2 may suppress contraction of colonic smooth muscle by upregulating expression of SK3 and inhibiting Ca2+ influx in rats.