Shedding light on DNA: Combined with the addition of a photosensitive cationic surfactant, we used light to control the single-chain higher-order structure of genomic duplex T4 DNA (166 kbp) in bulk solution and in cell-mimicking microcompartments. We demonstrate that this strategy can be used to encapsulate folded/unfolded genomic DNA inside phospholipid-coated cell-sized microdroplets with high efficiency and low DNA damage.