Genomic regions free of nucleosomes, which are hypersensitive to DNase I digestion, are known as DNase I hypersensitive sites (DHSs) and frequently contain cis-regulatory DNA elements. To investigate their prevalence and characteristics in maize (Zea mays), we developed high-resolution genome-wide DHS maps using a modified DNase-seq technique. Maize DHSs exhibit depletion of nucleosomes and low levels of DNA methylation and are enriched with conserved noncoding sequences (CNSs). We developed a protoplast-based transient transformation assay to assess the potential gene expression enhancer and/or promoter functions associated with DHSs, which showed that more than 80% of DHSs overlapping with CNSs showed an enhancer function. Strikingly, nearly 25% of maize DHSs were derived from transposable elements (TEs), including both class I and class II transposons. Interestingly, TE-derived DHSs (teDHSs) homologous to retrotransposons were enriched with sequences related to the intrinsic cis-regulatory elements within the long terminal repeats of retrotransposons. We demonstrate that more than 80% of teDHSs can drive transcription of a reporter gene in protoplast assays. These results reveal the widespread occurrence of TE-derived cis-regulatory sequences and suggest that teDHSs play a major role in transcriptional regulation in maize.