AbstractThe carbon efficiency of storage lipid biosynthesis from imported sucrose in green Brassicaceae seeds is proposed to be enhanced by the PRK/Rubisco shunt, in which ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts outside the context of the Calvin–Benson–Bassham cycle to recycle CO2 molecules released during fatty acid synthesis. This pathway utilizes metabolites generated by the nonoxidative steps of the pentose phosphate pathway. Photosynthesis provides energy for reactions such as the phosphorylation of ribulose 5-phosphate by phosphoribulokinase (PRK). Here, we show that loss of PRK in Arabidopsis thaliana (Arabidopsis) blocks photoautotrophic growth and is seedling-lethal. However, seeds containing prk embryos develop normally, allowing us to use genetics to assess the importance of the PRK/Rubisco shunt. Compared with nonmutant siblings, prk embryos produce one-third less lipids—a greater reduction than expected from simply blocking the proposed PRK/Rubisco shunt. However, developing prk seeds are also chlorotic and have elevated starch contents compared with their siblings, indicative of secondary effects. Overexpressing PRK did not increase embryo lipid content, but metabolite profiling suggested that Rubisco activity becomes limiting. Overall, our findings show that the PRK/Rubisco shunt is tightly integrated into the carbon metabolism of green Arabidopsis seeds, and that its manipulation affects seed glycolysis, starch metabolism, and photosynthesis.