Highlights 1. ORF3a was successfully transfected into SW480 cells, and its expression was localized in the cytoplasm of SW480 cells. 2. ORF3a inhibited SW480 cell proliferation, invasion and migration, induced their apoptosis, and blocked them in the G1 phase. 3. ORF3a inhibited colorectal tumor growth and accelerated cancer cell death in vivo. 4. Our study provides new insights into the use of viruses in anti-tumor regimens in colorectal cancer.