Embryonic stem (ES) cells exhibit general characteristics of open chromatin, a state that may be necessary for ES cells to efficiently self-renew while remaining poised for differentiation. Histone H3K4 and H3K9 trimethylation associate as a general rule, with open and silenced chromatin, respectively, for ES cell pluripotency maintenance. However, how histone modifications are regulated to maintain open chromatin in ES cells remains largely unknown. Here, we demonstrate that trithorax protein Ash2l, homologue of the Drosophila Ash2 (absent, small, homeotic-2) protein, is a key regulator of open chromatin in ES cells. Consistent with Ash2l being a core subunit of mixed lineage leukemia methyltransferase complex, RNAi knockdown of Ash2l was sufficient to reduce H3K4 methylation levels and drive ES cells to a silenced chromatin state with high H3K9 trimethylation. Genome-wide ChIP-seq analysis indicated that Ash2l is recruited to target loci through two distinct modes and enriched at a family of genes implicated in open chromatin regulation, including chromatin remodeler Cdh7, transcription factor c-Myc, and H3K9 demethylase Kdm4c. Our results underscore the importance of Ash2l in open chromatin regulation and provide insight into how the open chromatin landscape is maintained in ES cells.