DNA supercoiling refers to the amount of twist in a particular DNA strand, which determines the amount of strain on it. A given strand may be "positively supercoiled" or "negatively supercoiled" (more or less tightly wound). The amount of a strand's supercoiling affects a number of biological processes, such as compacting DNA and regulating access to the genetic code (which strongly affects DNA metabolism and possibly gene expression). Certain enzymes, such as topoisomerases, change the amount of DNA supercoiling to facilitate functions such as DNA replication and transcription. The amount of supercoiling in a given strand is described by a mathematical formula that compares it to a reference state known as "relaxed B-form" DNA.

In a "relaxed" double-helical segment of B-DNA, the two strands twist around the helical axis once every 10.4–10.5 base pairs of sequence. Adding or subtracting twists, as some enzymes do, imposes strain. If a DNA segment under twist strain is closed into a circle by joining its two ends, and then allowed to move freely, it takes on different shape, such as a figure-eight. This shape is referred to as a supercoil. (The noun form "supercoil" is often used when describing DNA topology.)

The DNA of most organisms is usually negatively supercoiled. It becomes temporarily positively supercoiled when it is being replicated or transcribed. These processes are inhibited (regulated) if it is not promptly relaxed. The simplest shape of a supercoil is a figure eight; a circular DNA strand assumes this shape to accommodate more or few helical twists. The two lobes of the figure eight will appear rotated either clockwise or counterclockwise with respect to one another, depending on whether the helix is over- or underwound. For each additional helical twist being accommodated, the lobes will show one more rotation about their axis.

Lobal contortions of a circular DNA, such as the rotation of the figure-eight lobes above, are referred to as writhe. The above example illustrates that twist and writhe are interconvertible. Supercoiling can be represented mathematically by the sum of twist and writhe. The twist is the number of helical turns in the DNA and the writhe is the number of times the double helix crosses over on itself (these are the supercoils). Extra helical twists are positive and lead to positive supercoiling, while subtractive twisting causes negative supercoiling. Many topoisomerase enzymes sense supercoiling and either generate or dissipate it as they change DNA topology.

In part because chromosomes may be very large, segments in the middle may act as if their ends are anchored. As a result, they may be unable to distribute excess twist to the rest of the chromosome or to absorb twist to recover from underwinding—the segments may become supercoiled, in other words. In response to supercoiling, they will assume an amount of writhe, just as if their ends were joined.

Supercoiled DNA forms two structures; a plectoneme or a toroid, or a combination of both. A negatively supercoiled DNA molecule will produce either a one-start left-handed helix, the toroid, or a two-start right-handed helix with terminal loops, the plectoneme. Plectonemes are typically more common in nature, and this is the shape most bacterial plasmids will take. For larger molecules it is common for hybrid structures to form – a loop on a toroid can extend into a plectoneme. If all the loops on a toroid extend then it becomes a branch point in the plectonemic structure. DNA supercoiling is important for DNA packaging within all cells, and seems to also play a role in gene expression.

Based on the properties of intercalating molecules, i.e. fluorescing upon binding to DNA and unwinding of DNA base-pairs, in 2016, a single-molecule technique has been introduced to directly visualize individual plectonemes along supercoiled DNA which would further allow to study the interactions of DNA processing proteins with supercoiled DNA. In that study, Sytox Orange (an intercalating dye) was used to induce supercoiling on surface tethered DNA molecules.

Using this assay, it was found that the DNA sequence encodes for the position of plectonemic supercoils. Furthermore, DNA supercoils were found to be enriched at the transcription start sites in prokaryotes.