Focal adhesion kinase (FAK), which plays a pivotal role in mediating cell proliferation, survival and migration, is frequently overexpressed in human colon cancer. In the present study, we utilized the short hairpin RNA (shRNA) to knock down the expression of FAK in SW480 human colon cancer cells in vitro. Furthermore, nude mice bearing human colon carcinoma SW480 were established and treated with plasmids encoding FAK shRNA encapsulated in DOTAP: Chol cationic liposome through tail vein injection. Tumor growth and potential side effect were observed during the treatment. Assessments of angiogenesis, cell proliferation, and apoptosis were performed by using immunohistochemistry against CD31, proliferating cell nuclear antigen (PCNA) and TUNEL assays, respectively. The results indicated that DOTAP: Chol could efficiently deliver the therapeutic plasmids systemically to tumor xenografts, resulting in suppression of tumor growth. Treatment with plasmid encoding FAK-targeted shRNA reduced mean tumor volume by approximately 86% compared with control groups (p < 0.01), accompanied with angiogenesis inhibition (p < 0.05), tumor cell proliferation suppression (p < 0.05) and apoptosis induction (p < 0.05). Taken together, our data demonstrated that shRNA-mediated silencing of FAK might be a potential therapeutic approach against human colon carcinoma.