X inactivation is controlled by Xist and its antisense gene, Tsix, neither of which encodes a protein. Xist is essential for X inactivation to occur in cis, and its differential expression is a key event in the initiation of X inactivation. Xist and Tsix are imprinted in the extraembryonic tissues of mouse embryos so that they are expressed from the paternal and maternal X, respectively, resulting in the preferential inactivation of the paternal X. Targeted disruption of Tsix causes ectopic expression of Xist, suggesting that Tsix negatively regulates Xist in cis. However, the molecular mechanism of this antisense regulation remains unknown. Here, we demonstrate that Tsix transcriptionally silences Xist in both embryonic and extraembryonic tissues of mouse embryos. Moreover, we show that disruption of Tsix impairs establishment of repressive epigenetic modifications and chromatin structure at the Xist locus. We propose that Tsix silences Xist through modification of the chromatin structure.