Protection of the germline from somatic differentiation programs is crucial for germ cell development. In many animals, whose germline development relies on the maternally inherited germ plasm, such protection in particular at early stages of embryogenesis is achieved by maternally localized global transcriptional repressors, such as PIE-1 of Caenorhabditis elegans, Pgc of Drosophila melanogaster and Pem of ascidians. However, zygotic gene expression starts in later germline cells eventually and mechanisms by which somatic gene expression is selectively kept under repression in the transcriptionally active cells are poorly understood. By using the ascidian species Halocynthia roretzi, we found that H3K27me3, a repressive transcription-related chromatin mark, became enriched in germline cells starting at the 64-cell stage when Pem protein level and its contribution to transcriptional repression decrease. Interestingly, inhibition of H3K27me3 together with Pem knockdown resulted in ectopic expression in germline cells of muscle developmental genes Muscle actin (MA4) and Snail, and of Clone 22 (which is expressed in all somatic but not germline cells), but not of other tissue-specific genes such as the notochord gene Brachyury, the nerve cord marker ETR-1 and a heart precursor gene Mesp, at the 110-cell stage. Importantly, these ectopically expressed genes are normally expressed in the germline sister cells (B7.5), the last somatic lineage separated from the germline. Also, the ectopic expression of MA4 was dependent on a maternally localized muscle determinant Macho-1. Taken together, we propose that H3K27me3 may be responsible for selective transcriptional repression for somatic genes in later germline cells in Halocynthia embryos and that the preferential repression of germline sister-lineage genes may be related to the mechanism of germline segregation in ascidian embryos, where the germline is segregated progressively by successive asymmetric cell divisions during cell cleavage stages. Together with findings from C. elegans and D. melanogaster, our data for this urochordate animal support the proposal for a mechanism, conserved widely throughout the animal kingdom, where germline transcriptional repression is mediated initially by maternally localized factors and subsequently by a chromatin-based mechanism.