C₄ carbon fixation is one of three biochemical mechanisms, along with C₃ and CAM photosynthesis, used in carbon fixation. It is named for the 4-carbon molecule present in the first product of carbon fixation in the small subset of plants known as C₄ plants, in contrast to the 3-carbon molecule products in C₃ plants.

C₄ fixation is an elaboration of the more common C₃ carbon fixation and is believed to have evolved more recently. C₄ and CAM overcome the tendency of the enzyme RuBisCO to wastefully fix oxygen rather than carbon dioxide in what is called photorespiration. This is achieved by using a more efficient enzyme to fix CO₂ in mesophyll cells and shuttling this fixed carbon via malate or asparate to bundle-sheath cells. In these bundle-sheath cells, RuBisCO is isolated from atmospheric oxygen and saturated with the CO₂ released by decarboxylation of the malate or oxaloacetate. These additional steps, however, require more energy in the form of ATP. Because of this extra energy requirement, C₄ plants are able to more efficiently fix carbon in only certain conditions, with the more common C₃ pathway being more efficient in other conditions.

In biology, carbon fixation is the reduction of inorganic carbon (carbon dioxide) to organic compounds by living organisms. The most prominent example is photosynthesis. Organisms that grow by fixing carbon are called autotrophs—plants for example. Heterotrophs, like animals, are organisms that grow using the carbon fixed by autotrophs. Fixed carbon, reduced carbon, and organic carbon all mean organic compounds.

Photosynthesis uses energy from sunlight to drive an autotrophic carbon fixation pathway.

Oxygenic photosynthesis is used by the chief primary producers—plants, algae, and cyanobacteria. They contain the pigment chlorophyll, and use the Calvin cycle to fix carbon autotrophically.

Somewhere between 3.5 and 2.3 billion years ago, cyanobacteria evolved oxygenic photosynthesis. The process works like this:

The essential innovation is the first step, the dissociation of water into electrons, protons, and free oxygen. This allows the use of water, one of the most abundant substances on Earth, as an electron donor—as a source of reducing power. The release of free oxygen is a side-effect of enormous consequence. The first step uses the energy of sunlight to oxidize water to O₂, and, ultimately, to produce ATP