Mono-methylation of Histone H3 Lysine 4 (H3K4me1) is a prominent mark of enhancers in animal cells, yet its role in enhancer function is still unclear. Here we provide evidence suggesting a role for this histone mark and its writer proteins in mediating long-range chromatin interactions at enhancers. We first show that DNA looping between Sox2 and its distal enhancer depends on Mll3 and Mll4 (Mll3/4), the histone methyltransferases responsible for H3K4me1, in mouse embryonic stem (mES) cells. We then demonstrate that chromatin interactions originated from active enhancers in mES cells are abrogated in the absence of Mll3/4 proteins. Additionally, we find that during mES cell differentiation, H3K4me1 modification and long-range chromatin interactions formed at the lineage-specific enhancers are severely reduced in the absence of Mll3/4, along with transcription of lineage-specific genes.  Finally, we show that the cohesin complex could associate with H3K4me1-marked mononucleosome in vitro, and depletion of the cohesin complex results in decreased chromatin interactions at enhancers without affecting H3K4me1 status. Taken together, our results support a model that H3K4 methyltransferases mediate chromatin interactions at enhancers, suggesting an active role for H3K4me1 in higher order chromatin organization in mammalian cells.