Aquaporin (AQP)-5 is an essential member of AQP family involved in the tumorigenesis of various malignant tumors. However, its role in the angiogenesis of colorectal cancer is unclear and requires further investigation. In this study, a pRNA-H1.1 vector containing the short hairpin RNA (shRNA) targeting AQP5 mRNA was constructed to inhibit the endogenous expression of AQP5 in human umbilical vein endothelial cells (HUVECs). We found that the AQP5-silenced HUVECs acquired decreased proliferation, migration and tube formation ability. AQP5 shRNA also inhibited the enzyme activity of matrix metalloprotease (MMP)-9 in HUVECs without affecting the MMP-2. Further, two colorectal cancer cell lines (HT29 and HCT116) stably transfected with scrambled or AQP5 shRNA were established. The expression and secretion of vascular endothelial growth factor (VEGF)-A (a pro-angiogenic factor) in colorectal cancer cells were downregulated by AQP5 shRNA. HUVECs cultured in low-VEGF conditioned media (CM) obtained from cancer cells developed less vessel-like tubes and had decreased proliferation and migration. The growth and angiogenesis of xenograft tumors were suppressed when the endogenous AQP5 in HT29 cells was knocked down. Tumor samples were additionally collected from patients with colorectal cancer to analyze the expression of AQP5. The immunofluorescence data indicated that AQP5 was expressed in both inner cancer areas and CD31-positive vessels. Taken together, our study suggests AQP5 as a novel anti-angiogenesis target for colorectal cancer.