Thereis a paradox that theexperimentallydefined regulatory elements span hundreds of base pairs (~270 bp) whereas bioinformatics generated motifs only span ~6-14 bp in the genome.To figurethisout, we developed a method termed as MAE-seq (Massive Active Enhancers by Sequencing) to experimentally identify functional regulatory elements at a 25-bpscale.In this study,19,514 active enhancers are identifiedin mouse embryonic stem cells. Out of these, 49.25% overlap with known ones and 50.75%are de novoones without any annotation at EnhancerAtlas 2.0.Validation results show that these 25-bp sequences could act as a functional unit, which shows identical or similar expression patterns as the previous defined larger elements. Our results also demonstrate that some of the predicted short motifs need the flanking sequences at both ends to work as a functional unit.Additionally, we examined the functions of some novel elements that can up-regulate gene activities. By integrating with Hi-C data, 61.86% novel elements may have distal association with different targeted genes. These imply the “multiple elements-one target gene” pattern is more prevalent than expected. As an example, Cdh1 gene has been found interacting with one novel and two known regulatory elements in mESCs. CRISPR-Cas9 approach is introduced to investigate their biological effects and the results indicate these distal associations are hierarchically coordinated to regulate Cdh1 gene expression and mESCs proliferation. Our study provides an experimental approach to refine the regulatory elements at 25-bpresolution andit has the potential tounscramble the underlying information across the genome.