Epithelial–mesenchymal transition (EMT) is an essential step for cancer metastasis. MicroRNAs (miRNAs) are small non-coding RNAs that regulate target-mRNAs post-transcriptionally. The expression and function of miRNAs in EMT of HT-29 colonic cells remain elusive. This study looks at expression of miRNAs in EMT and explores the effects of miRNAs on EMT in HT-29 cell line. HT-29 was treated with TGF β to establish an EMT model, in which a collection of miRNAs was dynamically regulated by real-time PCR (qPCR) analysis. Among them, miR-21 and miR-27 were significantly upregulated, while miR-22, miR-26, miR-30, miR-181, miR-200b, miR-200c and miR-214 were markedly downregulated. MiRNA-inhibitors were used to knockdown miRNAs in HT-29 and EMT markers were determined by qPCR to monitor the effects of miRNAs on EMT process. Results showed that miR-22 could not alter the expression of EMT markers, while knockdown of miR-200b could significantly increase that of epithelial markers, N-cadherin, Vimentin, α-Sma and Twist1 and decrease that of mesenchymal marker, E-cadherin. Bioinformatic analysis and Western blot showed that ZEB1 was directly suppressed by miR-200b. In conclusion, miRNAs are dynamically regulated in TGF β-induced EMT of HT-29 and miR-200b was essential for EMT by suppressing the expression of ZEB1 in HT-29.