C₃ carbon fixation is the most common of three metabolic pathways for carbon fixation in photosynthesis, the other two being 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.^[1] 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,^[2] 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.^[3] 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), contributing to more ¹³C depletion seen in C₃ plants compared to C₄ plants especially since the C₄ pathway uses PEP carboxylase in addition to Rubisco.^[4]

Not all C3 carbon fixation pathways operate at the same efficiency.

Bamboos and the related rice have an improved C3 efficiency. This improvement might be due to its ability to recapture CO₂ produced during photorespiration, a behavior termed "carbon refixation". These plants achieve refixation by growing chloroplast extensions called "stromules" around the stroma in mesophyll cells, so that any photorespired CO₂ from the mitochondria has to pass through the RuBisCO-filled chloroplast.^[5]

Refixation is also performed by a wide variety of plants. The common approach involving growing a bigger bundle sheath leads down to C2 photosynthesis.^[6]

C3 carbon fixation is prone to photorespiration (PR) during dehydration, accumulating toxic glycolate products. In the 2000s scientists used computer simulation combined with an optimization algorithm to figure out what parts of the metabolic pathway may be tuned to improve photosynthesis. According to simulation, improving glycolate metabolism would help significantly to reduce photorespiration.^[7][8]

Instead of optimizing specific enzymes on the PR pathway for glycolate degradation, South et al. decided to bypass PR altogether. In 2019, they transferred Chlamydomonas reinhardtii glycolate dehydrogenase and Cucurbita maxima malate synthase into the chloroplast of tobacco (a C₃ model organism). These enzymes, plus the chloroplast's own, create a catabolic cycle: acetyl-CoA combines with glyoxylate to form malate, which is then split into pyruvate and CO₂; the former in turn splits into acetyl-CoA and CO₂. By forgoing all transport among organelles, all the CO₂ released will go into increasing the CO₂ concentration in the chloroplast, helping with refixation. The end result is 24% more biomass. An alternative using E. coli glycerate pathway produced a smaller improvement of 13%. They are now working on moving this optimization into other C₃ crops like wheat.^[9]