Abstract Purpose: The expression of LIM and SH3 protein 1 (LASP1) was upregulated in colorectal cancer cases, thereby contributing to the aggressive phenotypes of colorectal cancer cells. However, we still cannot decipher the underlying molecular mechanism associated with colorectal cancer metastasis. Experimental Design: In this study, IHC was performed to investigate the expression of proteins in human colorectal cancer tissues. Western blot analysis was used to assess the LASP1-induced signal pathway. Two-dimensional difference gel electrophoresis was performed to screen LASP1-modulated proteins and uncover the molecular mechanism of LASP1. TGFβ was used to induce an epithelial–mesenchymal transition (EMT). Results: LASP1 expression was correlated with the mesenchymal marker vimentin and was inversely correlated with epithelial markers, namely, E-cadherin and β-catenin, in clinical colorectal cancer samples. The gain- and loss-of-function assay showed that LASP1 induces EMT-like phenotypes in vitro and in vivo. S100A4, identified as a LASP1-modulated protein, was upregulated by LASP1. Moreover, it is frequently coexpressed with LASP1 in colorectal cancer. S100A4 was required for EMT, and an increased cell invasiveness of colorectal cancer cell is induced by LASP1. Furthermore, the stimulation of TGFβ resulted in an activated Smad pathway that increased the expression of LASP1 and S100A4. The depletion of LASP1 or S100A4 expression inhibited the TGFβ signaling pathway. Moreover, it significantly weakened the proinvasive effects of TGFβ on colorectal cancer cells. Conclusion: These findings elucidate the central role of LASP1 in the TGFβ-mediated EMT process and suggest a potential target for the clinical intervention in patients with advanced colorectal cancer. Clin Cancer Res; 20(22); 5835–47. ©2014 AACR.