JUNB is an activating protein-1 transcription factor and is important in the control of cell growth, differentiation and neoplastic transformation. To understand the role of JUNB in human hepatocellular carcinoma (HCC), we examined 30 HCCs and their paired non-cancerous tissues by real-time quantitative reverse transcription-polymerase chain reaction and immunohistochemical analysis for the expression of JUNB. The results showed that JUNB was underexpressed in most of the cancerous tissues (73.3%). Mutational analysis of the entire gene, and methylation analysis of CpG sites at the promoter area of JUNB, showed no specific changes between cancerous and paired non-cancerous tissues. Our results suggest that the down-regulated JUNB expression in HCC was due to an unknown mechanism, and not to methylation of the CpG site at the promoter or mutation in the coding areas. We further analyzed the relationship between JUNB, P16 and cyclin D1 expression. Our results showed that JUNB and P16 produced similar expression patterns, however, inverse expression patterns were found between JUNB and cyclin D1 in most of the HCC tissues. We also found a discrepancy between the expression of JUNB and cyclin D1 in some of the HCC tissue. From these results, we suggest that two pathways (JUNB-related and JUNB-unrelated) may be involved in the development of HCC.