Previous evidence showed β1, 3-N-acetylglucosaminyltransferase 8 (β3GnT8), which can extend polylactosamine on N-glycans, to be highly expressed in some cancer cell lines and tissues, indicating roles in tumorigenesis. However, so far, the function of β3GnT8 in laryngeal carcinoma has not been characterized. To test any contribution, Hep-2 cells were stably transfected with sense or interference vectors to establish cell lines that overexpressed or were deficient in β3GnT8. Here we showed that cell proliferation was increased in β3GnT8 overexpressed cells but decreased in β3GnT8 knockdown cells using MTT. Furthermore, we demonstrated that change in β3GnT8 expression had significant effects on tumor growth in nude mice.We further provided data suggesting that overexpression of β3GnT8 enhanced the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) at both the mRNA and protein levels, associated with shedding of tissue inhibitors of metalloproteinase TIMP-2. In addition, it caused increased production of transforming growth factor beta 1 (TGF-β1), whereas β3GnT8 gene knockdown caused the reverse effect. The results may indicate a novel mechanism by which effects of β3GnT8 in regulating cellular proliferation are mediated, at least in partvia targeting MMPs/TIMPs and TGF-β1 in laryngeal carcinoma Hep-2 cells. The finding may lay a foundation for further investigations into the β3GnT8 as a potential target for therapy of laryngeal carcinoma.