MicroRNA (miRNA) is associated with tumor cell proliferation, migration and invasion. Studies have shown that miRNAs are closely related to the occurrence and development of colorectal cancer (CRC), but the mechanisms deserve further investigation. In this study, we aim to explore the role of miR-363 on CRC tumorigenesis. Using CRC cell lines, we tested the expression of miR-363 by using RT-PCR, and miR-363 effect on cell behavior was test by using CCK-8 assay, wound-healing assay and cell invasion assay, and western blotting. Luciferase reporter assay and western blot confirmed that E2F3 was the target gene for miR-363. We further examined the effect of E2F3 on the regulation of miR-363 on cell behavior through knockdown of E2F3. Western blot and RT-PCR assay showed that miR-363 inhibited the expression of E2F3 in HCT-116 and SW480 cell. MiR-363 overexpression or E2F3 knockdown inhibited cell proliferation, migration and invasion of CRC. This study demonstrated that miR-363 is able to suppress cell proliferation, migration and invasion by negative regulating E2F3 in CRC cells, and inhibits tumor growthin vivo.