A cofactor is any substance that needs to be present in addition to an enzyme to catalyse a certain reaction. (However, more or less ubiquitous substances such as water do not qualify.)

Some cofactors are inorganic, such as the metal atoms zinc, iron, and copper in certain forms. Others, such as most vitamins, are organic.

Cofactors generally are either bound tightly to active sites, or may bind loosely with the substrate. When a cofactor is an organic substance that directly participates as a substrate in the reaction, it is called a coenzyme.