Objective：The aim of the study was to investigate the effect of 17β-estradiol（E2）on the expression of RhoA/ROCK pathway in rat colon smooth muscle cells（SMC）. Methods：Sprague-Dawley rat colonic SMCs were isolated and cultured in vitro. Double immunofluorescence staining was used to observe the expression of ROCK1/ERα and α-actin. After stimulation with E2 at different concentrations and different times，the expression of RhoA and ROCK1 were detected by reverse transcription real-time quantitative PCR（qRT）-PCR and Western blot. After stimulation with different concentrations of ROCK inhibitor Y27632 and 17β-estradiol，the expression of ROCK downstream protein was detected by Western blot. The effects of estrogen receptor（ER）inhibitor ICI182780，bovine serum albumin-bound（BSA-E2），ERα selective agonist propyl pyrazole triol（PPT）and ERβ selective agonist diarylpropiolnitrile（DPN）on RhoA/ROCK pathway expression were observed. The ERα and ERβ siRNAs were constructed，and the effects of E2 stimulation on the expression of RhoA and ROCK1 in SMCs were observed. Results：Double immunofluorescence staining showed that ROCK1 and α-SMA co-expressed in rat colon SMC. Treatment with 50 nmol/L E2 for 24 h could significantly inhibit the expression of RhoA and ROCK1（P < 0.05）. ROCK inhibitor Y27632 inhibited the expression of downstream proteins p-MYPT1，p-CPI17 and p-MLC in a concentration-dependent manner. The expressions of RhoA and ROCK1 in E2 group and PPT group were significantly lower than those in other groups（P < 0.05）. The expressions of RhoA /ROCK1 in ERα siRNA group were higher than those in control group（P < 0.05）. Conclusion：ROCK1 is localized in rat colonic SMCs，and E2 inhibits the RhoA/ROCK pathway in an ERα-dependant manner．