DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling, we used biotinylated trimethylpsoralen as a DNA structure probe to show that the human genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF insulator protein-binding sites. Underwound domains are transcriptionally active and enriched in topoisomerase I, 'open' chromatin fibers and DNase I sites, but they are depleted of topoisomerase II. Furthermore, DNA supercoiling affects additional levels of chromatin compaction as underwound domains are cytologically decondensed, topologically constrained and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation, providing an evolutionary purpose for clustering genes along chromosomes.