Previous evidence showed β1, 3-N-acetylglucosaminyltransferase 8 (β3GnT8), which can extendpolylactosamine on N-glycans, to be highly expressed in some cancer cell lines and tissues, indicating roles intumorigenesis. However, so far, the function of β3GnT8 in laryngeal carcinoma has not been characterized. Totest any contribution, Hep-2 cells were stably transfected with sense or interference vectors to establish cell linesthat overexpressed or were deficient in β3GnT8. Here we showed that cell proliferation was increased in β3GnT8overexpressed cells but decreased in β3GnT8 knockdown cells using MTT. Furthermore, we demonstrated thatchange in β3GnT8 expression had significant effects on tumor growth in nude mice.We further provided datasuggesting that overexpression of β3GnT8 enhanced the expression of matrix metalloproteinase-2 (MMP-2) andmatrix metalloproteinase-9 (MMP-9) at both the mRNA and protein levels, associated with shedding of tissueinhibitors of metalloproteinase TIMP-2. In addition, it caused increased production of transforming growthfactor beta 1 (TGF-β1), whereas β3GnT8 gene knockdown caused the reverse effect. The results may indicate anovel mechanism by which effects of β3GnT8 in regulating cellular proliferation are mediated, at least in partviatargeting MMPs/TIMPs and TGF-β1 in laryngeal carcinoma Hep-2 cells. The finding may lay a foundation forfurther investigations into the β3GnT8 as a potential target for therapy of laryngeal carcinoma.