Inherent and acquired chemoresistance reduce the effectiveness of irinotecan in the treatment of metastatic colorectal cancer (CRC). However, the molecular mechanisms underlying this resistance process are still unclear. Twist1 is one of the master transcription factors of epithelial-mesenchymal transition (EMT). Our previous study indicated that Twist1 is overexpressed in colon cancer tissues, and demonstrated that Twist1 plays a crucial role in the chemoresistance of CRC. In the present study, we further investigated how Twist1 contribute to acquired resistance to irinotecan in colon cancer. The irinotecan-resistant cells were established by gradual adaptation of increasing irinotecan concentrations in LoVo cells, named LoVo/CPT-11R cells. Results showed that cell viabilities to different anticancer drugs were markedly increased in LoVo/CPT-11R cells compared to LoVo cells. Moreover, LoVo/CPT-11R cells displayed EMT, CSC-like cellular morphology and relative biomarkers were also significantly increased. In addition, overexpressed Twist1 LoVo cells were established by lentivirus transfection assay, named LoVo/Twist1 cells. Results showed that the LoVo/Twist1 cells perform a distinctly decreased sensitivity to irinotecan, downregulated expression of E-cadherin, upregulated expression of cluster of differentiation 44 (CD44), and a significant enhancement of invasion and migration potential by regulation of MMP2 compared with control cells. In contrast, the inhibition of Twist1 transfected with siRNA could enhance the irinotecan sensitivity in LoVo/CPT-11R cells and downregulate the expression of vimentin and CD44. Our data provide evidence that EMT and CSC-like phenotype induced by Twist1 contribute to acquire resistance to irinotecan and enhanced migration and invasion in colon cancer.