Aberrant genome-wide hypomethylation has been thought to be related to tumorigenesis. However, its mechanism and implications in hepatocellular carcinogenesis remain to be elucidated. Samples of hepatoma (hepatocellular carcinoma, HCC) and paired non-HCC liver tissues were obtained from 17 HCC patients. Normal liver tissues obtained from three individuals were used as controls. Compared with the paired non-HCC liver tissues, genome-wide 5-methylcytosine content in HCC was reduced in all of the tested HCC samples (P < 0.001). Conversely, genome-wide 5-methylcytosine content did not significantly differ among normal, noncirrhotic, and cirrhotic liver tissues. Moreover, the degree of reduced DNA methylation was related to late histopathological HCC grade (P = 0.005) and large tumor size (P = 0.079). Compared with the paired non-HCC liver tissues, expression of DNA methyltransferases DNMT-1, DNMT-3A, and DNMT-3B and the DNA methyltransferase-like gene, DNMT-2, was up-regulated in 53, 41, 59, and 47% of the HCC samples, respectively. Surprisingly, small amounts of LINE-1 retrotransposon transcripts were detected in HCC and non-HCC as well as normal liver tissues, and the expression levels were not significantly different in HCC compared with the paired non-HCC or normal liver tissues. Of interest, the 3' ends of these LINE-1 transcripts were truncated. Our findings suggest that genome-wide hypomethylation in HCC is a continuing process that persists throughout the lifetime of the tumor cells rather than a historical event occurring in precancer stages or in cell origins for HCC. Up-regulation of DNA methyltransferases might simply be a result of increased cell proliferation in cancer. In addition, our results did not support the hypothesis of activation of transposable elements in HCC via genome-wide hypomethylation.