Background: ACTL8 is a member of the CT antigens. There are only few studies on the role of ACTL8 in malignant tumors. The aim of this study is to investigate the expression and clinical significance of ACTL8 protein in colorectal cancer (CRC). Materials and methods: Human CRC tissues and cell lines, and paired adjacent non-tumor tissues and human intestinal epithelial cell lines were obtained to evaluate the expression of ACTL8. The association between protein expression of ACTL8 and clinicopathological parameters and prognosis of CRC patients was examined. The biological functions of ACTL8 in the invasion and metastasis of CRC were determined by wound healing and transwell invasion assays after silencing of ACTL8 in CRC cell lines. The potential target genes of ACTL8 were also identified by quantitative reverse transcription PCR and Western blotting after silencing of ACTL8 in CRC cell lines. Results: It was found that ACTL8 was upregulated in human CRC tissues and cell lines. The expression of ACTL8 was positively associated with poor differentiation, invasion and metastasis, postoperative infection, and poor prognosis, but negatively associated with proximal margin length. In addition, silencing of ACTL8 significantly decreased the capacity of invasion and migration in HT29 and SW620 CRC cell lines. Moreover, silencing of ACTL8 significantly decreased the expression of TRIM29 in HT29 and SW620 CRC cell lines. Conclusion: These results suggest that ACTL8 plays a key role in the invasion and metastasis of CRC, and TRIM29 may be involved in the ACTL8-mediated poor prognosis of CRC.