PSII assembly requires auxiliary factors, including Psb28. Although the absence of Psb28 in cyanobacteria has little effect on PSII assembly, we show here that the Chlamydomonas (Chlamydomonas reinhardtii) psb28 null mutant is severely impaired in PSII assembly, showing drastically reduced PSII supercomplexes, dimers, and monomers, while overaccumulating early PSII assembly intermediates reaction center II (RCII), CP43mod, and D1mod. The mutant had less PSI and more cytochrome b6f complex, its thylakoids were organized mainly as monolayers, and it had a distorted chloroplast morphology. Complexome profiling of the psb28 mutant revealed that THYLAKOID ENRICHED FRACTION 5 (TEF5), the homolog of Arabidopsis (Arabidopsis thaliana) PHOTOSYSTEM B PROTEIN 33/LIGHT HARVESTING-LIKE 8, comigrated particularly with RCII. TEF5 also interacted with PSI. A Chlamydomonas tef5 null mutant was severely impaired in PSII assembly and overaccumulated RCII and CP43mod. RC47 was not detectable in the light-grown tef5 mutant. Our data suggest a possible role for TEF5 in RCII photoprotection or maturation. Both the psb28 and tef5 mutants exhibited decreased synthesis of CP47 and PsbH, suggesting negative feedback regulation possibly exerted by the accumulation of RCII and/or CP43mod in both mutants. The strong effects of missing auxiliary factors on PSII assembly in Chlamydomonas suggest a more effective protein quality control system in this alga than in land plants and cyanobacteria.