Metastasis often occurs in colorectal cancer (CRC) patients and is the main difficulty in cancer treatment. The upregulation of poly-N-acetyllactosamine-related glycosylation is found in CRC patients and is associated with progression and metastasis in cancer. β-1,4-Galactosyltransferase III (B4GALT3) is an enzyme responsible for poly-N-acetyllactosamine synthesis, and therefore, we investigated its expression in CRC patients. We found that B4GALT3 negatively correlated with poorly differentiated histology (P< 0.001), advanced stages (P= 0.0052), regional lymph node metastasis (P= 0.0018) and distant metastasis (P= 0.0463) in CRC patients. B4GALT3 overexpression in CRC cells suppressed cell migration, invasion and adhesion, whereas B4GALT3 knockdown enhanced malignant cell phenotypes. The β1 integrin-blocking antibody reversed the B4GALT3-mediated increase in cell invasion. B4GALT3 expression altered glycosylation on the N-glycan of β1 integrin probably through changes in poly-N-acetyllactosamine expression. Furthermore, more activated β1 integrin along with the activation of its downstream signaling transduction were found in B4GALT3 knockdown cells, whereas overexpression of B4GALT3 suppressed the expression of active β1 integrin and inhibited its downstream signaling. Our results suggest that B4GALT3 is negatively associated with CRC metastasis and suppresses cell invasiveness through inhibiting activation of β1 integrin.