Abstract: Objective To test the effect of CDC42 (a member of Rho family of small GTPases) knockdown mediated by a CDC42 short-hairpin RNA (shRNA) on the morphology of colorectal cancer SW480 cells in vitro. Methods Four CDC42 siRNA fragments targeting CDC42 were designed and the most efficient siRNA for CDC42 knockdown was selected to construct the shRNA vector for transfection of colorectal cancer SW480 cells. The interference efficiency in the stably transfected cells (sw480. shCDC) was detected using real-time PCR and Western blotting, and the morphological changes of the transfected cells were observed. Results Western blotting result showed that siCDC42-3 was the most efficient fragment for CDC42 knockdown, which caused CDC42 knockdown by over 50%. DNA sequencing confirmed successful construction of the CDC42 shRNA vector. Transfection of the cells with the vector significantly reduced CDC42 expressions at both the mRNA and protein levels. The transfected cells exhibited reduced filopodia and cell size with smooth cell margins. Conclusion shRNA-mediated CDC42 knockdown can reduce the cytoskeleton dynamics of colorectal cancer cells to lower their invasiveness. This shRNA construct facilitates further study of the role of CDC42 in the tumorigenesis and progression of colorectal cancer.