KITENIN promotes cancer cell migration and invasion in vitro and cancer metastasis in mouse cancer models, including colon and head and neck cancers. The purposes of this study were to observe the effect of KITENIN on tumor cell behaviors of human hepatocellular carcinoma (HCC) cells and to evaluate its expression in human HCC tissues. To functionally characterize KITENIN in human HCC, we depleted its expression in human HCC cell lines, HepG2 and Huh7, by using small interfering RNA (siRNA). Invasion and proliferation assays were performed. The activator protein-1 (AP-1) transcriptional activity and expression of AP-1 target genes were evaluated by AP-1 luciferase reporter assay and RT-PCR. The contribution of mitogen-activated protein kinase (MAPK) cascade signaling involved in AP-1 activation was assessed by Western blotting. We evaluated the expression of KITENIN and AP-1 target genes at mRNA levels by RT-PCR in human HCC tissues and paired normal hepatic mucosa of the same patients taken by surgery. Knockdown of KITENIN in HepG2 and Huh7 cells resulted in a significant reduction of tumor cell invasion. The tumor cell proliferation was significantly decreased in the KITENIN knocked down Huh7 cells compared to the negative control. The mRNA expressions of MMP-3 and COX-2 were decreased in KITENIN knocked down Huh7 cells. The mRNA expression of MMP-1 was decreased in KITENIN knocked down HepG2 cells. The AP-1 transcriptional activity in Huh7 cells was significantly decreased by knockdown of KITENIN. The JNK and ERK1/2 phosphorylations were decreased in KITENIN knocked down HepG2 and the p38 phosphorylation was decreased in KITENIN knocked down Huh7 cells. The mRNA expressions of KITENIN, MMP-1, and MMP-3 were significantly upregulated in human HCC tissues compared to paired normal mucosa. These results indicate that KITENIN is associated with activation of AP-1 target genes via MAPK cascades signaling in human HCC progression.