C₃ carbon fixation is the most common of three metabolic pathways for carbon fixation in photosynthesis, along with C₄ and CAM. This process converts carbon dioxide and ribulose bisphosphate (RuBP, a 5-carbon sugar) into two molecules of 3-phosphoglycerate through the following reaction:

CO₂ + H₂O + RuBP → (2) 3-phosphoglycerate

This reaction was first discovered by Melvin Calvin, Andrew Benson and James Bassham in 1950. C₃ carbon fixation occurs in all plants as the first step of the Calvin–Benson cycle. (In C₄ and CAM plants, carbon dioxide is drawn out of malate and into this reaction rather than directly from the air.)

Plants that survive solely on C₃ fixation (C₃ plants) tend to thrive in areas where sunlight intensity is moderate, temperatures are moderate, carbon dioxide concentrations are around 200 ppm or higher, and groundwater is plentiful. The C₃ plants, originating during Mesozoic and Paleozoic eras, predate the C₄ plants and still represent approximately 95% of Earth's plant biomass, including important food crops such as rice, wheat, soybeans and barley.

C₃ plants cannot grow in very hot areas at today's atmospheric CO₂ level (significantly depleted during hundreds of millions of years from above 5000 ppm) because RuBisCO incorporates more oxygen into RuBP as temperatures increase. This leads to photorespiration (also known as the oxidative photosynthetic carbon cycle, or C2 photosynthesis), which leads to a net loss of carbon and nitrogen from the plant and can therefore limit growth.

C₃ plants lose up to 97% of the water taken up through their roots by transpiration. In dry areas, C₃ plants shut their stomata to reduce water loss, but this stops CO₂ from entering the leaves and therefore reduces the concentration of CO₂ in the leaves. This lowers the CO₂:O₂ ratio and therefore also increases photorespiration. C₄ and CAM plants have adaptations that allow them to survive in hot and dry areas, and they can therefore out-compete C₃ plants in these areas.

The isotopic signature of C₃ plants shows higher degree of ¹³C depletion than the C₄ plants, due to variation in fractionation of carbon isotopes in oxygenic photosynthesis across plant types. Specifically, C₃ plants do not have PEP carboxylase like C₄ plants, allowing them to only utilize ribulose-1,5-bisphosphate carboxylase (Rubisco) to fix CO₂ through the Calvin cycle. The enzyme Rubisco largely discriminates against carbon isotopes, evolving to only bind to ¹²C isotope compared to ¹³C (the heavier isotope), attributing to why there's a low ¹³C depletion seen in C₃ plants compared to C₄ plants especially since the C₄ pathway uses PEP carboxylase in addition to Rubisco.