Although Notch1 expression has been associated with progression or prognosis in various tumors, the role of Notch1 in hepatocellular carcinoma (HCC) remains unknown. This study sought to investigate the clinicopathological and prognostic relevance of Notch1 expression in HCC as well as the underlying mechanisms responsible. HCC tissues were stained with an anti-Notch1 antibody. The invasion capacities of cells were measured using Transwell cell culture chambers. Reverse transcription PCR and/or western blot were used to evaluate the expression levels of Notch1, matrix metalloproteinase (MMP)-2, and MMP-9. Notch1 expression was downregulated by RNA interference. The activity of MMP-2/MMP-9 was quantified by enzyme-linked immunosorbent assay, and cellular apoptosis was analyzed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Notch1 expression was mainly localized within the cytoplasm and at the cell membrane. High Notch1 expression correlated with tumor size, tumor grade, metastasis, venous invasion, and American Joint Committee on Cancer TNM stage (P < 0.05), and patients with high levels of Notch1 expression were at a significantly increased risk for shortened survival time (P < 0.05). In vitro, the downregulation of Notch1 expression decreased the invasion capacity of HCC cells via the regulation of MMP-2 and MMP-9. The results of the MTT assay showed that downregulation of Notch1 did not affect HCC cell viability. Notch1 may represent a novel candidate marker for patient prognosis as well a molecular target for HCC therapy.