Hexadecameric form I Rubisco, consisting of eight large (RbcL) and eight small (RbcS) subunits, is the most abundant enzyme on earth. Extensive efforts to engineer an improved Rubisco to speed up its catalytic efficiency and ultimately to increase agricultural productivity have been undertaken. However, genetic manipulation of hexadecameric Rubisco has been hampered by its difficulty to fold and assemble correctly in foreign hosts or in vitro. Here we reconstituted Synechococcus sp. PCC6301 Rubisco with the chaperonin system and assembly factors from cyanobacteria and Arabidopsis thaliana (At) in vitro. Rubisco holoenzyme was produced in the presence of either Rubisco accumulation factor 1(Raf1) from cyanobacteria alone, or AtRaf1 and bundle-sheath defective-2 (AtBsd2) from Arabidopsis. RbcL released from GroEL is assembly capable in the presence of ATP and AtBsd2 functions downstream of AtRaf1. Cryo-EM structures of RbcL₈-AtRaf1₈, RbcL₈-AtRaf1₄-AtBsd2₈, and RbcL₈ revealed that the interactions between RbcL-AtRaf1 are looser than those between prokaryotic RbcL-Raf1, with AtRaf1 tilting 7 degrees farther away from RbcL. AtBsd2 stabilizes the flexible regions of RbcL, including the N-, C-terminus, the 60s loop and loop 6. Using these data and combined with previous findings, we propose models for the biogenesis pathways of prokaryotic and eukaryotic Rubisco.