The histone demethylase LSD1, a component of the CoREST (corepressor for element 1-silencing transcription factor) corepressor complex, plays an important role in the downregulation of gene expression during development. However, the activities of LSD1 in mediating short-time-scale gene expression changes have not been well understood. To reveal the mechanisms underlying these two distinct functions of LSD1, we performed genome-wide mapping and cellular localization studies of LSD1 and its dimethylated histone 3 lysine 4 (substrate H3K4me2) in mouse embryonic stem cells (ES cells). Our results showed an extensive overlap between the LSD1 and H3K4me2 genomic regions and a correlation between the genomic levels of LSD1/H3K4me2 and gene expression, including many highly expressed ES cell genes. LSD1 is recruited to the chromatin of cells in the G(1)/S/G(2) phases and is displaced from the chromatin of M-phase cells, suggesting that LSD1 or H3K4me2 alternatively occupies LSD1 genomic regions during cell cycle progression. LSD1 knockdown by RNA interference or its displacement from the chromatin by antineoplastic agents caused an increase in the levels of a subset of LSD1 target genes. Taken together, these results suggest that cell cycle-dependent association and dissociation of LSD1 with chromatin mediates short-time-scale gene expression changes during embryonic stem cell cycle progression.