Recent studies have demonstrated that natural antisense transcripts, which are complementary sequences to messenger RNA, have important cellular functions such as the stabilization and silencing of mRNA. However, the possible contribution of antisense transcripts in hepatocellular carcinoma (HCC) development has not been described. Therefore, we simultaneously investigated the sense and antisense transcripts of HCC and non-cancerous tissues to explore the possible contribution of antisense transcripts to HCC progression. RNA was prepared from 15 HCV-associated HCCs and from 6 corresponding non-cancerous tissues and was subjected to expression profile analysis of sense and antisense transcripts using a human custom microarray. Differential expression of 161 sense and 25 antisense transcripts was observed with more than 2-fold between HCC and non-cancerous tissue (p<0.001). The expression of the sense and antisense transcripts was used to cluster cancer and non-cancerous tissues, and the cancer and non-cancerous tissues were found to be clearly separated into different clusters. Additionally, the sense and antisense expression profiles were analyzed with regard to HCC differentiation (p<0.001), resulting in 71 sense and 43 antisense transcripts. These unique transcripts did not overlap with those found in the discrimination of HCC from non-cancerous tissues. When the HCC tissues were clustered by transcript expression, the antisense transcripts resulted in clustering of HCC that was consistent with grouping based on histology. These findings strongly indicate that the antisense transcripts together with the sense transcripts are involved in liver tumorigenesis.