Linker histone H1 associates with nucleosomes, facilitating folding and packaging of DNA into higher order chromatin structure. With 11 variants in mammals, histone H1 is the most divergent histone class. Histone H1 variants are differentially expressed during development and cellular differentiation, and regulate specific gene expression in vivo. Ample studies have established the role of linker histone H1 in chromatin compaction and gene expression regulation; however, its role in diseases, such as cancer, remain understudied. In this study, we explore the role of H1 in ovarian cancer, one of the most devastating gynecological cancers due to its poor prognosis and difficulty in early diagnosis. Although mutations of genes responsible for cell proliferation, differentiation and survival have been found in ovarian cancers, ample evidence also suggests an important role of epigenetic changes in the disease occurrence and progression. Because epigenetic changes do not alter DNA sequence and can be reversed or reprogrammed, they offer an attractive avenue for therapeutic intervention in cancer treatment. Using quantitative RT-PCR assays, we systematically examined the expression of 7 H1 genes in 33 human epithelial ovarian tumors. By clustering analysis, we found that ovarian malignant adenocarcinomas and benign adenomas exhibited characteristic expression patterns. We demonstrate that expression profiling of 7 H1 genes in tumor samples discriminates adenocarcinomas vs. adenomas with high accuracy. These findings indicate that the expression of H1 variants is exquisitely regulated and may serve as potential epigenetic biomarkers for ovarian cancer. To further investigate the role of H1 subtypes in ovarian cancer cells, we employ an over-expression approach to test the function of H1 subtypes in an ovarian cancer cell line OVCAR-3. We found that histone H1.3 over-expression significantly suppresses the growth and colony formation of OVCAR-3 cells. Gene expression arrays identified many genes affected by H1.3 over-expression, and oncogene H19 is among the genes most dramatically repressed by H1.3 over-expression. Over-expression of several other H1 subtypes does not lead to significant reduction of H19 expression, suggesting a specific effect by H1.3. Consistently, knockdown of H1.3 increases H19 expression. Furthermore, increased expression of H1.3 leads to accumulation of H1.3 as well as increased DNA methylation at the regulatory regions of H19. Finally we identified a synergistic effect of H1.3 over-expression and H19 knockdown on inhibition of ovarian cancer cell growth. These results establish oncogene H19 as a direct target of histone H1.3, identify a novel role of H1 variants in ovarian cancer mediated through regulating oncogene H19 expression, and may offer new approaches for ovarian cancer therapeutics.